JPH01225483A - Recombinant plasmid - Google Patents

Abstract

PURPOSE:To obtain a microorganism in Klebsiella in which nifL gene is deficient and a promoter is integrated in the upper area of the nifA gene, thus having high nitrogen-fixing ability even in the presence of ammonia. CONSTITUTION:The nifAL operon originating from K. oxytoca, which has been cloned on the plasmid is processed to deactivate nifL gene. At the same time, a recombinant plasmid in which a specific promoter is integrated in the upper area of the nifA gene is introduced into a microorganism having nitrogen- fixing ability to replace with the homologous gene on the chromosome to obtain a microorganism in which the nifL gene on the chromosome is deficient and a specific promoter is integrated in the upper area of the nifA gene on the chromosome. The microorganism is used to develop the nifA gene on the chromosome structurally whereby the inhibition of nitrogen fixation in the presence of ammonia is markedly reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a microorganism useful for nitrogen fixation in plants and a recombinant plasmid that can be used to construct the microorganism. Therefore, the present invention is useful in the agricultural field.

BACKGROUND OF THE INVENTION Research on enhancing nitrogen fixation ability in plants has been widely conducted, and many studies have been conducted at the genetic level.

Nitrogen fixation genes (hereinafter referred to as nif genes) are used in microorganisms belonging to the genus Klebsiella, especially K.
Research on iae) is progressing. In Figure 1, pne
The structure of the nif gene of Umoniae is schematically shown.

The 17 nif genes have a full-length 23 K b (Ki
l.

base pair), and a nitrogenase precursor is generated from n1fK and D. nif Q
, B, V.

N and E are involved in the production of FaMo cofactors.

n1fH encodes the nitrogenase reductase precursor, and nif'vl is required for its activation. n1fF encodes flavotoxin as an electron carrier, and n1fJ encodes flavotoxin reductase. The black and white circles in FIG. 1 represent promoters, and the arrows attached to them represent the direction of transcription (mRNA production) from each operon. n
The products of ifA and L regulate the expression of the promoters (black circles) of all other nif genes, and the nifA product acts as an activator, but 0
In the presence of 2 or when a sufficient N source such as ammonia is present, the nifL product acts as a repressor. This is a very sensible adjustment to prevent waste. Since nitrogenase is quickly inactivated by oxygen,
This eliminates the waste of synthesizing the nitrogenase system in the presence of 02. Furthermore, when a fixed N source exists, the fixation of N is stopped to prevent wasted energy.

The nifLA operon itself is regulated by a higher order control mechanism of the entire nitrogen metabolic system as shown in FIG. That is, there are ntrB and C genes that form the same operon as the glutamine synthetase gene (glnA), which is important as an enzyme that incorporates ammonia into amino acids, and the ntrc and ntrB products cooperate to form nifL.
In addition to the A operon, it regulates the expression of genes related to active transport (argTr, dhuA) for absorbing and assimilating N-rich amino acids such as arginine and histidine. Under conditions where an intracellular N source is sufficient, ntrBC products act as repressors for these genes of the nitrogen metabolic system, and under conditions where an N source is deficient, they act as activators. In this way, the expression of the nif gene system for aerial nitrogen fixation is strictly regulated in two stages by ntrBC and nifLA, and this reflects the fact that the nitrogen fixation reaction requires a large amount of energy. Therefore, the nitrogen fixation system does not develop unless there is a particular shortage of nitrogen sources.

In this way, the genes of the nitrogen metabolic system, including the nitrogen fixation system, are under common control, and for this to happen, some common structure must exist on the promoter side. Already, pneumoniae's n1fH,'n1f
Nucleotide sequences of various nif genes, including D, are being published, but in particular, the sequences of the promoter portions of all nif operons of K. pneumoniae have been determined, and a comparison of their structures shows that
CTGGCAC, -1 at the -24 site upstream of the transcription start site
There is a TTGCA consensus sequence (common sequence) at two sites. Furthermore, these sequences are glnA, dhuA
.

It is also common to the promoters of nitrogen metabolic system genes such as argTr, but -35 of the normal E. coli promoter
The consensus sequence present at the and -10 positions is completely different. Note that the ntrA gene in Figure 2 is nifL.
It encodes the σ factor of RNA polymerase, which is commonly required for the expression of A and other nitrogen metabolic system promoters, and is used to recognize the common promoter of nitrogen metabolic system genes.

Bacteria that associate with the rhizosphere of non-leguminous plants and fix nitrogen have recently attracted attention. Rice.

These include Azospirillum, Klebsiella, and Enterobacter in the rhizosphere of corn, wheat, grasses, etc. In Japan, the ability of microorganisms to fix nitrogen in the rhizosphere of rice has attracted attention, and it has been reported that this ability varies markedly depending on the quality of the rice. From its rhizosphere, Talebsiella oxytophora (
Klebsiellaoxytoca) N G 13
, Azospiri
Nitrogen-fixing bacteria such as L. llum lipoferum) COC8 have been isolated.

The present inventors introduced oxytoc into nitrogen-fixing bacteria in the rice rhizosphere.
The nif gene of aNG13 was used to induce pneumonia.
n1fKDH of e was detected as a probe, and all of its ni
By cloning the f gene group (ni f Q-J, 23 Kb) in E. coli and expressing nitrogen-fixing activity in E. coli, we would like to succeed in growing E. coli in a nitrogen-free medium (see Figure 3). Agric, Biol
.

Chem, 49.1469 (1985)).

As a result of analyzing this gene group by restriction enzyme digestion, the gene structure was shown in Figure 1.
Although it is roughly the same as the one shown in the figure, some restriction enzyme cleavage sites are different, and the homology between the base sequences of the nif gene group in both figures was estimated to be about 96%.

In addition, a multicopy plasmid p carrying all nif genes of oxytoca, and a composite plasmid pN carrying BR322.
The nitrogenase activity (acetylene reducing ability) of E. coli having OW25 was about one-tenth that of K, oxytoca NG l 3, and nitrogen fixation activity was not improved simply by increasing the copy number of the nif gene. This is thought to be because the physiological conditions within E. coli are not necessarily optimal for the expression of the complex nitrogen fixation system genes. Furthermore, since nitrogen fixation reactions require a large amount of energy, energy supply may be the rate limiting factor.

Another way to improve the nitrogen fixing ability of rhizosphere bacteria is to use ni
This involves cutting out the fA gene, connecting it to a constitutive promoter, and expressing it. This should release the ammonia-induced rift and cause KN fixation to occur even in the presence of ammonia. The inventors o
xytoca NG 13 nifA gene to pACY
pNOW? is linked to the C184 tetracycline resistance gene promoter. I created A. This was introduced into various bacteria, and K, oxytoca NG 13 (pN
OW? A), K.

pneumoniae UNF714 (pNOW
7A) and ε, coli KO60 (pNOW25.
pNOW? When the nitrogen fixing activity of A) was measured, it showed that in the presence of ammonia, the nitrogen fixing ability was about 1 to 20% higher than that in the absence of ammonia [Japan Agricultural Chemistry Society 1981 Conference Lecture IH-10 ( 1984), A.
gric.

8io1. Che+++,, 50.1539~1
544. (1986)].

pNOW? In the absence of A, nitrogen fixation activity is completely suppressed in the presence of ammonia, so although the effect of constitutive expression of the ni fA gene was certainly observed, the expression of nitrogen fixation activity was not sufficient. Ta. In this system, since the nifL gene is present on the chromosome, it is thought that the inhibitory effect was exerted in the presence of ammonia.

Klebsiella oxytoca NG 13
For the nucleotide sequence of the nifLA operon,
Mat, Gen, Genet,
(1986) 205:253-259.

Problems to be Solved by the Invention It is desired to develop a method for improving the nitrogen fixing ability of rhizosphere bacteria. As mentioned above (K, activated gene n in oxytoca
Introduction of a multicopy plasmid that constitutively expresses ifA enhances nitrogen fixation ability to some extent, but in reality, it has an inhibitory effect in the presence of ammonia, and the nitrogen fixation ability is reduced to about 20% in the presence of ammonia. .

Means for Solving the Problems According to the present invention, cloned onto a plasmid,
oxytoca-derived nifLA operon was engineered to produce n
The ifL gene is inactivated and a recombinant plasmid with a specific promoter incorporated upstream of the nifA gene is introduced into a microorganism that belongs to K. oxytoca and has nitrogen fixation ability, and is recombined with the homologous gene on the chromosome. This provides a microorganism in which the nifL gene on the chromosome is deleted and a specific promoter is incorporated upstream of the nifA gene on the chromosome. Using this microorganism, inhibition of nitrogen fixation ability in the presence of ammonia can be significantly reduced by constitutively expressing the nifA gene on the chromosome.

In this way, by introducing a recombinant plasmid containing a constitutive promoter upstream of the nifA gene into a microorganism that has modified the nitrogen-fixing gene on its chromosome, it is possible to fully express nitrogen-fixing ability even in the presence of ammonia. The present inventors have discovered that it is possible to obtain microorganisms that can produce the desired results, leading to the completion of the present invention.

The present invention will be explained in detail below.

The present invention provides the nifL gene deleted with a marker, n
A recombinant plasmid containing the ifA gene and a promoter integrated upstream of the nifA gene is provided.

The recombinant plasmid of the present invention is introduced into a microorganism that belongs to Klebsiella species and has nitrogen-fixing ability, and by recombining with a homologous gene on the chromosome,
A microorganism lacking the L gene can be obtained. This microorganism can constitutively express the nifA gene on its chromosome, and inhibition of nitrogen fixation ability in the presence of ammonia is significantly reduced.

A specific example of the recombinant plasmid of the present invention is a plasmid named plasmid pNOY9cm.

pNOY9cm is constructed as follows (see Figure 4).

(1) Construction of plasmid pNoys-Tet Plasmid pBR322 was digested with restriction enzymes, E3afI and 5Sp.
1.6 K by agarose electrophoresis.
Collect the DNA fragment b (hereinafter referred to as fragment a).

On the other hand, plasmid pNOY8 is cut with restriction enzyme C1aI, and a DNA fragment of about 8.0 Kb is recovered by agarose electrophoresis. This DN using S1 nuclease
Blunt the ends of the A fragment.

The precipitate recovered by extraction with phenol and ether and precipitation with ethanol is treated with alkaline phosphatase, and a DNA fragment of about 8, QKb (hereinafter referred to as fragment) from which blunt-end phosphates have been removed is recovered by agarose electrophoresis. .

Fragment a and fragment A were ligated using T41J gauze, the ligated product was used to transform Escherichia coli JM105, and a plasmid pNOY8- of approximately 9.6 Kb was obtained from the transformant.
Get Te t.

(2) Construction of plasmid pNOY9 Plasmid pNOY8 was cut with restriction enzyme PvuII,
Approximately L 6 Kb of DNA was determined by agarose electrophoresis.
Collect the fragments. phosphorylated with this DNA fragment) (i
ndI [I linker is ligated using T4 ligase, Kpn I and HindllJ are added to the ligated product, a digestion reaction is performed, and a DNA fragment of approximately 620 bp (fragment 1) is recovered by agarose electrophoresis.

On the other hand, pNOY8-Tet was digested with Kpn I and Nco I, and approximately 800 bp of D
NA fragment and approximately 8.8 Kb DNA fragment (fragment 2
). The approximately 800 bp DNA fragment is further pvu[
A 176 bl) DNA fragment (fragment 3) is recovered by agarose electrophoresis.

Additionally, plasmids pYEJOO1 were transformed into Hind■ and 5
Digested with spI and analyzed by agarose electrophoresis to approximately 3
A DNA fragment (fragment 4) of 00 bp (calculated 276 bp) is recovered.

The four DNA fragments obtained above were ligated using T4 ligase, the ligated product was used to transform Escherichia coli JMI O5, and the plasmid pNO was extracted from the transformant according to a standard method.
Collect Y9.

(3) Plasmid 1) Construction of NOV9C+n After cutting plasmid pNOY9 with restriction enzyme Sca I, remove the terminal phosphate using alkaline phosphatase, and collect a DNA fragment of about 9.9 Kb by agarose electrophoresis. .

On the other hand, limit plasmid pBR328! i! The protein is cleaved with HHA, the protein is inactivated with phenol, and the protein is precipitated with ethanol in the same manner as described above. This precipitate contains dATP and dCT.
P, dGTP, dTTP and T4 DNA polymerase are applied to make the DNA ends blunt. This blunted DNA fragment and the DNA fragment obtained above were ligated using T4 ligase, and the ligated product was used to infect E. coli JM.
About 11.35K was transformed from the transformant.
Obtain the plasmid pNOY9cm of b.

The present invention also provides a microorganism into which the above recombinant plasmid has been introduced.

Any microorganism can be used as long as the nif gene of the recombinant plasmid can be expressed efficiently in the presence of ammonia. A specifically preferred strain is Klebsiellaoxytoca NG
13 can be given. Klebsiel 1aoxyto
Klebsiella oxytoca NG 13 using Klebsiella oxytoca NG 13 and the above recombinant plasmid.
The transformed strain obtained by transforming oxytoca
Rl 6 is FERM BP-17 to FIKEN respectively.
40 and 1741 on February 17, 1988.

By further introducing a plasmid incorporating only the nifA gene into the recombinant microorganism of the present invention, inhibition of nitrogen fixation ability in the presence of ammonia can be largely avoided.

As the plasmid incorporating the nifA gene, any plasmid that can express the nifA gene in microorganisms can be used. Specific - for example,
Examples include plasmid pNOA102. plasmid p
NOA 102 is specified by the restriction map shown in FIG. 5, and is manufactured by the method described below.

The plasmid pNOA1 was cut with the restriction enzyme Tthllll, the ends were blunted with S1 nuclease in the same manner as above, and the size was reduced to about 1.8 K b by agarose electrophoresis.
Collect the DNA fragment (fragment A).

On the other hand, plasmid pBR328 was transformed with restriction enzyme PvuI [,
It was cleaved with EcoRV, the terminal phosphate was removed with alkaline phosphatase, and about 3.
A 3 Kb DNA fragment (fragment opening) is recovered.

Both fragments are ligated with T4 ligase. The conjugate was used to transform E. coli JMI 05, and approximately 5.2
K b plasmid pNOA101 is recovered.

Plasmid pNOA101 was cut with the restriction enzyme Sca, and a DNA of approximately 5.2 Kb was obtained by agarose electrophoresis.
Collect A fragment. pBR328 was cut with the restriction enzyme HhaI, and a D of approximately 1.6 K b was obtained by agarose electrophoresis.
Collect NA fragments.

Both DNA fragments are ligated using T4 ligase, E. coli JM105 is transformed using the ligated product, and plasmid pNOA102 is recovered from the transformed strain.

K, plasmid p'NOA to oxytoca R16
Introduction of 102; Transformation method with Ca Cj'2 (Norgard,
14. V.

Keem, K., Monahan, J.J., Gene
3.279 (1978)), o
xytoca R16 (pNOA102) is FERM
It was deposited as BP-1742 at the Institute of Fine Technology on February 17, 1988.

The reaction conditions for the above recombinant technique are as follows.

The digestion reaction of DNA with restriction enzymes is usually 0.1 to 20
of DNA at 2-200mM (preferably 10-40mM
) of Tri 5-HCl (pH 6,0-9.5 preferably pH 7,0-8.0), 0-200mM Na
Cl, M of 2-20mM (preferably 5-10mM)
g Restriction enzyme 0.1-10 in the reaction solution containing C12
The reaction is carried out using 0 units (preferably 1 to 3 units per 1 μg of DNA) at 20 to 70°C (the optimum temperature varies depending on the restriction enzyme used) for 15 minutes to 24 hours.

The reaction is usually stopped by heating at 55 to 75° C. for 5 to 30 minutes, but a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

Purification of DNA fragments generated by restriction enzyme digestion was performed using low melting point agarose gel electrophoresis (LGT method) [L, Wieslander: analytical biochemistry (^analytical B
iochemistry) 98.305 (1979
)) and polyacrylamide gel electrophoresis [A, M, Maxam et al.: Proceedings of the National Academy of Sciences x (Proc, Natl, Acad, S
ci,).

USA 74.560 (1977) E, etc.

The DNA fragment binding reaction is carried out using 2 to 200 mM (preferably 10 to 40 mM) Tri 5-HCI (pH 6,1
~9.5, preferably pH 7.0-8.0).

2-20mM (preferably 5-10mM) MgClx
, 0.1-10mM (preferably 0.5-2.0mM
) of ATP, I ~50mM (preferably 5~lom
M) in the reaction solution containing dithiothreitol, T4DN
Using 0.3 to 10 units of A ligase, the reaction is carried out at 1 to 37°C (preferably 3 to 20°C) for 15 minutes to 72 hours (preferably 2 to 20 hours).

Note that the conditions for the digestion reaction and binding reaction may be other than those described here, and in the case of commercially available products, they can be carried out according to the conditions indicated by the supplier.

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Jorgard et al. (Gene
, 3.279 (1978)) into E. coli or Klebsiella oxytoca.

Isolation of recombinant plasmid DNA from Escherichia coli can be carried out using the method of Humphreys et al. (Biochim, Biophys, Acta,
383.457 (1975) 3 or Birnboim et al.'s method [HoC, Bi
rnboim) et al.: Nucleic Acids Res, 1
513 (1979)].

After digesting plasmid DNA with 1 to 10 types of restriction enzymes, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis. Furthermore, when it is necessary to determine the base sequence of DNA, the method of Sanger et al. [Proceedings of the National Academy of Sciences] using M13 phage vector is used.
of Science (Proc, Natl, Acad)
, Sci.).

74, 5463-5467 (1977)].

The nitrogen fixation activity in the present invention is determined by a method using acetylene reduction (Uozumi et al., Agric, Bi
ol.

Chem, 50.1539-1544 (1986)
].

Examples of the present invention will be shown below.

Example 1)'pNOY8-TetcDIIi 2■ of Nibrasmid pBR322 and 10 units of restriction enzyme Bal■ (manufactured by Zenshuzo Co., Ltd., 1009A) were mixed with 204 Bal I buffer (composition: 20mM Tris'-HCl, 7
The mixture was placed in mM MgC (lx, 7mM 2-mercaptoethanol, pH 8,5) and reacted at 37°C for 1 hour. Furthermore, restriction enzyme 5spi Nippon Gene, 311
-01102) 10 units and 20 units of Ssp I buffer (
Composition: 100mM NaCl, 10mM Tris-H
Cl, 10mM g Cj! 2. 10mM
2-Mercabutoeta/-r.

After reacting at 37°C for 1 hour, the reaction solution was subjected to agarose electrophoresis (Sharp et al.
at, Biochemistry (Bioche+n1str)
Approximately 1.6 K by ydan: 3055 (1973)
DNA fragment of b (Chen.

C,W,et al,, Anal,Biochem,
, 101: 339 (1980)). This is called fragment (a).

On the other hand, plasmid pNOY8 [Mo1. Gen, Gen
etc.

504 C1aI buffer (composition: 10mM Tri
s-HCI, 7mM MgCj! z, 50mM NaC
j! .

pH 8,0) and reacted at 37°C for 1 hour,
Approximately 8. OK
Approximately 10 μg of DNA fragment b was recovered. To this DNA fragment, 15 units of 81 nuclease (manufactured by Zenshuzo Co., Ltd., 241OA) and 31 nuclease buffer (composition: 30mM sodium acetate, 100mM NaCl, 1mM ZnS) were added.
04. pH 4,6) to make it 100 μf2, 22
The ends were blunted by reacting at ℃ for 2 hours.

Add the same volume of phenol (Molecular) to this reaction solution.
Cloning, Co1d Spring H
Arbor Laboratory (1982)
.

[prepared according to the method described in p. 43g] was added and shaken at room temperature for 2 to 3 minutes to inactivate the protein. Centrifugation (15
After removing the aqueous layer and extracting the remaining phenol with ether, add 2.5 times the volume of ethanol and sodium acetate with a final concentration of 300 mM to precipitate the mixture with ethanol.The following ethanol precipitation method is used. ). Precipitate with 204 IM Tris-HCI
(pH 9), add 2 units of alkaline phosphatase (manufactured by Zenshuzo Co., Ltd., 211OA), and heat at 65°C for 4 hours.
After reacting for 0 minutes, agarose electrophoresis was performed to remove blunt-end phosphates. Five DNA fragments of Q K b were recovered. This is called a fragment (b).

Fragment (a) 0.5 ttg, fragment (b) 0.5Ag and 1 unit of T4 ligase (manufactured by Zenshuzo Co., Ltd., 211OA) in 40g of glue gauze buffer [composition: 0.66M Tr
i s-HCI (pH 7,6), 50mM Mg (1
2,50m, M dithiothreitol, '10mM
The mixture was added to ATP:I and reacted at 4°C for 16 hours.

Escherichia coli JM 105 (Ame
rsham) was transformed (Norgard, MJ
,, et al,, Gene3, 279 (197
8)) Birnboim (Birnb) from L, transformant
oim) et al.'s method (Nucleic Ac1ds
Res, 7: 1513 (1979)) plasmid pNOY8-''rat was obtained.

Example 2 Construction of plasmid pNOY9Cm: 10 gg of plasmid FpNOY8 (71) and restriction enzyme Pv
u■ (manufactured by Zenshuzo Co., Ltd., 1076A) 10 units and 304 PvuII buffer (composition: 100mM Tris-HC (
1,7mM MgC1x, 60mM NaC1゜7mM
2-Mercaptoethanol, pH 7,5)
After reacting at 7°C for 2 hours, DNA fragment 5R of about 1.6 Kb was recovered from the reaction solution by agarose electrophoresis. This is followed by a phosphorylated Hind linker (12mar
, New England Biolabs IM
.

#1098) lx, 10 mM ATP, 40 μQ of ligase buffer, and T4'J gauze 2 were added, and the mixture was reacted at 4°C for 16 hours, and then heated at 65°C for 15 minutes to inactivate the enzyme. Two units of KpnI (manufactured by Zenshuzo Co., Ltd., 1068) were added to this reaction solution, and the mixture was reacted at 37° C. for 1 hour. Add to this NaCj at a final concentration of 50mM! and H
After adding 2 units of indIII and reacting at 37°C for 1 hour, agarose electrophoresis was performed, and approximately 620 bp of D
One piece of NA fragment was recovered.

This is called fragment 1.

Plasmid pNOY8-Tet 5g and Kpn I5
ML and 20 d of Kpn I buffer (6mM Tris
-HCj was added to 2.6mM Mg (J!x, 6mM 2-mercaptoethanol, pH 7,5) and reacted at 37°C for 1 hour. Add Ncol (manufactured by Shuzo Co., Ltd., 1160A
), 5 units were added, the reaction was carried out at 37° C. for 1 hour, and agarose electrophoresis was performed to recover about 2 μ of a DNA fragment of about 800 bp and about 2 Itg of a DNA fragment of about 8.8 Kb (fragment 2). About 800 bp fragment 1xr and PvuI[1
units and Pvu [buffer 20111, at 37°C for 1
The reaction mixture was allowed to react for several hours, and 176 bl) of DNA fragments were collected by agarose electrophoresis. This is called fragment 3.

Plasmid pYEJOO1 (Pharmacia Fine
Manufactured by Chemical Co., Lot Nα27-4928-01) 5J
tg, HindI[[5 units and 5sp15 units to 4
0Jdl of 5spI buffer solution, reacted at 37°C for 1 hour, and analyzed approximately 300bp DNA by agarose electrophoresis.
Approximately 3 cm of A fragment was recovered. This is called fragment 4.

Fragment 1. Fragment 2. Fragment 3. Approximately 0.5■ each of fragments 4
and 2 units of Tl gauze were placed in ligase buffer 404 and reacted at 4°C for 16 hours. Using the reaction solution, the conventional method (N
Orgard et al., Gene 3゜27
9 (1978)) according to Escherichia coli JMI 05 (Am
ersham), and the transformed strain was transformed using a conventional method (Birnboimetal, Nucleic).
Ac1ds Res, 7: 1513 (197
9) The plasmid was recovered according to ). This plasmid is called pNOY9.

Plasmid pNOY902 and Sca I (Bo
ehringer! , manufactured by Iannheim, #775
258) 2 units and 5 cal buffer solution (composition: 6
mMTriS-HCI, 150mM NaCl, 6mM
MgCl,.

6mM 2-mercaptoethanol, pH 7,
5> and reacted at 37° C. for 2 hours, and about 1 g of about 9.9 Kb (7) DNA fragment was obtained from the reaction solution by agarose electrophoresis.

This fragment was treated in the same manner as in Example 1, and a DNA fragment of about 9.9 Kb from which the terminal phosphate was removed was recovered by agarose electrophoresis. This is called fragment (I).

Plasmid pBR328 (manufactured by Zenshuzo Co., Ltd., #3050) 2
ttg and restriction enzyme Hh a I (Nippon G
ene, #319-00162) and 20 scraps of Hha buffer (composition: 5QmM Tris-HC).
j! .

5mM MgCl2.7mM 2-1 lkabutoethanol, pH 7,9) and reacted at 37°C for 1 hour, the same volume of phenol was added to the reaction solution, and the protein was inactivated by shaking at room temperature for 3 minutes. Centrifugation (15,OOOrp
After removing the aqueous layer and extracting the remaining phenol with ether, 2.5 times the volume of ethanol and sodium acetate at a final concentration of 300 mM were added to perform ethanol precipitation. The precipitate was dissolved in 204 TE buffer (Tri
5-HCI 10mM, EDTA 1mM, pH
7,5), dATP, dc at a final concentration of 0.1mM.
TP, dGTP and dTT, P and 2 units of T4
DNA polymerase and the same buffer (33mM Tr
Add i s -acetic acid (pH 7,9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, pH 7,91, and incubate at 37°C for 15 minutes.
The ends were blunted by reacting for a minute. The mixture was further heated at 70° C. for 5 minutes to inactivate T4 DNA polymerase. Ligase buffer containing ATP IQmM was added to this reaction solution.
About 1 inch of A12 and fragment (I) and 1 unit of Tl gauze were added and reacted at 4°C for 16 hours.

Using this reaction solution, E. coli JMI 05 was transformed according to a conventional method, and the plasmid was recovered according to a conventional method. This plasmid is called pNOY9cm.

Example 3 Construction of plasmid pNOY102; Plasmid pNOA 1 (Mol, Gen, G
enet, 1986205:253-259)
2■ and restriction enzyme TthllI (manufactured by Takara Shuzo Co., Ltd., 1
090) 2 units and 204 Tth1111 buffer (composition: 20mM Tris-HCC10mM Mg
C1*, 50mM NaCCIQmM 2-mercaptoethanol, pH 7,5) at 65°C.
The reaction mixture was allowed to react for several hours, and about one DNA fragment of about 1.8 Kb was recovered from the reaction solution by agarose electrophoresis. Add 1 unit of 81 nuclease and 31 nuclease buffer to this DNA fragment to yield 100 Il! 1 and reacted at 22°C for 2 hours to make the ends blunt. About 0.5 μ of a DNA fragment of about 1.8 Kb was recovered from this reaction solution by agarose electrophoresis.

This is called fragment (a).

Two units of plasmid pBR328 and two units of pvuII (manufactured by Takara Shuzo Co., Ltd., 1076) were added to 20m of PvuI [buffer (
10mM Tri5-HCj! , 7mM MgC1
x, 60mM NaCl, 7mM 2-mercaptoethanol, pH 7.5> and reacted at 37°C for 1 hour. Approximately 4.0% of the reaction solution was analyzed by agarose electrophoresis.
Approximately 1 μ of a 9 K b fragment was recovered.

This DNA fragment and EcoRV (manufactured by Takara Shuzo Co., Ltd.).

1042) and 1 unit in a 20d EcoRVll equilibrium solution (
10mM Tri5-HCl, 7mM MgCl
The mixture was placed in 150mM NaCl, 7mM 2-yt silcatoethanol, pH 7.5, and reacted at 37°C for 1 hour. LM Tris-HCj2 was added to this reaction solution.
(pH 9) buffer 2.54 and 1 unit of alkaline phosphatase were added, reacted at 37°C for 400 minutes, and subjected to agarose electrophoresis to remove blunt-end phosphates.
．． Approximately 0.5 μ of a 3 Kb DNA fragment was recovered. This is called a fragment (b).

Fragment (A) 0.5■, Fragment (B) 0.5 Cod, ATPIQ
One unit of mM and T4'J gauze was placed in 40ρ ligase buffer and reacted at 4°C for 16 hours.

E. coli JM105 was transformed using this reaction solution according to a conventional method, and the plasmid was recovered according to a conventional method. This plasmid is called pNOAlol.

1■ of plasmid pNOA101 and restriction enzyme 5caI
1 unit in 20ρ Sca I buffer at 37°C.
After 2 hours of reaction, approximately 5
．． Approximately 0.5 μ of a 2 Kb DNA fragment was recovered.

On the other hand, 2J1g of pBR328 and 2 units of restriction enzyme HhaI were added to 20m(7)Hhal buffer, reacted for 1 hour at 37°C, and 1■ DNA fragment of about 1.6 Kb was collected from the reaction solution by agarose electrophoresis. did.

Approximately 0.5 μm of the fragment derived from pNOA101, 1 g of the fragment derived from pBR328, 10 mM of ATP, and 1 unit of T4 ligase were added to 20 m glue gauze buffer and reacted at 4° C. for 16 hours. Using this reaction solution, E. coli JMI O5 was transformed according to a conventional method, and the plasmid was recovered according to a conventional method. This plasmid is called pNOA102.

・Example 4゜oxytoca NG using pNOY9c+n
Transformation of 13 and acquisition of recombinant strains: Klebsiella oxytoca (Klebsiella o
xytoca) NG13 strain into L-broth (10 g of Bactodributon).

Bakto yeast extract 5g, NaCji! 5g
, Glucose Ig, R20ll2) 3 in 10ml
Pre-cultured at 7°C overnight, 1 ml of Q was inoculated into 10 ml of pre-warmed L-broth, and cultured with shaking at 37°C. After 2 hours, when ODSSG = 0.25, cool on ice, 4°C, 3.50 Orpm, 10
Bacteria were collected by centrifugation for 1 minute. Washing buffer (0,1M
NaCl, 5mM MgCJ! z, 5mM Tri
Add 5 ml of 5-HCC pH 7,6) and suspend at 4°C.
Centrifugation was performed at 3.50 Orpm for 5 minutes to collect bacteria. This washing operation was repeated once more. Precipitate with 0°C CaClx solution (5mM Tri 5-HCj!).

100mM CaC1z, 250mM KCl, 5mM
Suspend in 4 ml of MgC1x, pH 7,6) and
It was left standing at ℃ for 1 hour. Centrifugation was performed at 3.50 Orpm for 5 hours, and the precipitate was suspended in the same CaCβ2 solution 2004.

To this, plasmid diluted with TE buffer or NOY9C
Approximately 1 inch of DNA was added, and the mixture was allowed to stand at 0°C for 1 hour, with occasional stirring. Heat stimulation was performed at 42°C for 2 minutes, and the mixture was allowed to stand at 0°C for 5 minutes to incorporate DNA. Furthermore, L-brot
1 ml of h was added and cultured at 37°C for 90 minutes. Add this to L-brot containing 25g/ml of chloramphenicol.
h agar plate medium and cultured at 37°C.

The obtained transformed strain was added to 5 ml of LB medium (1% Bactodributon, 0.5% Bacto yeast extract).

Subculturing was performed 8 times at 37° C. for 8 to 15 hours using Inokinilum size 2% (NaClO, 5%, pH 7.2). Ethidium bromide at 750 μg/m1 was added to this and cultured at the same temperature for 15 hours to remove the plasmid.

Spread the culture solution on an agar plate (composition: LB medium, agar 1.3%) containing tetracycline lO■/+++l,
The colonies that appear are divided into pNOY9cm and n on the chromosome.
It was selected as a strain that had undergone recombination due to homology with if LA. (■ or ■ in Figure 6) The agar plate containing 10 JLg/ml of tetracycline on which the above colonies appeared was transferred to the agar plate containing 10 JLg/ml of tetracycline and 25 μg/ml of oyohychloramphenicol (composition: LB Replicas were performed on culture medium + agar 1.3%), and tetracycline-resistant and chloramphenicol-sensitive (Tc'Cm') strains were selected. This strain is a recombinant strain that has a Tl if L-AC marker on its chromosome and is called R16.

Figure 6 shows the introduction of plasmid pNOY9cm into the K. oxytoca NG 13 chromosome and the resulting o
The structure of the chromosome introduction site of xytoca R16 is shown.

Example 5 Confirmation of recombinant strain R16 by Southern hybridization: 5 oxytoca NG 13 strains and R16 strain
ml Noshiichi broth (composition: Bactotryptone 10g, Bacto yeast extract 5g, NaCA 5g.

Glucose Ig, p) (7,2) was used to culture at 37°C for 8 hours, 1 ml of the culture solution was inoculated into 100 ml of Shiichigross, and cultured at 37°C for 8 hours. After bacterial collection, the bacterial cells were added to 50 mM Tris-HCl (p
The suspension was suspended in 10 ml of H8,0), 10 μl of lysozyme was added, and the mixture was left at 37° C. for 30 minutes to lyse the bacteria.

Add sodium dodecyl sulfate (SDS) to the lysis solution at a final concentration of 1%.
), then added the same volume of phenol, mixed well, left at 60°C for 1 hour, and centrifuged (10,00 rpm).
, 30 minutes) to obtain the supernatant.

Add the same volume of cold ethanol to the supernatant and generate chromosome D.
NA was taken up with a glass i and dissolved in 2 ml of TEI equilibrium solution. RNasa I in solution 50/m].

RN a s e T11 (add bcg/+nl 3
The mixture was left at 7°C for 60 minutes, and phenol 21T11 was added and stirred to inactivate the protein. This liquid is centrifuged (12,
000rpm.

The aqueous layer was removed, and the remaining phenol layer was extracted with ether. Approximately 2504 chromosomal DNA precipitates were thus obtained each.

1 piece each of NG13 and R16 chromosomal DNA.

5 each of plasmids pNOY8 and pNOY9cm
0 ng of each and 1 unit of each of the restriction enzymes Smal and 1 unit of Pvul were added to 204 Sma 1 units.
Buffer (composition 10mM Tris-H (J!.

7mM Mg(J!z, 20mM K(1,7mM2
-Mercaptoethanol, pH 8,0) and Pvu
I buffer (composition: 10mM Tris'H (J!, 7
mM MgC1z, 150mM KC#.

7mM 2-mercaptoethanol, pH 8,0), reacted at 37°C for 5 hours, heated at 65°C for 10 minutes, subjected the reaction solution to agarose horizontal gel electrophoresis, and denatured with alkaline gel (0,5M NaOH-1,5M N1a
After performing C1), the DNA fragments were transferred to a nitrocellulose filter and fixed on the filter at 80° C. for 2 hours under reduced pressure.

On the other hand, 5 units of pNOY8 and 5 units of SmaI and P
The DNA fragment was digested in the same manner as above using 5 units of vuI, and subjected to agaro gel electrophoresis to recover a DNA fragment of 3.3 Kb and 750 bp. Sma obtained in this way
25ng of Pvu l fragment (3,3Kb) and Pvu l fragment (7
50bp) 25ng and l(andomPrimed
DNA labeling kit (
Amersham cat.

The probe DNA was labeled with 32p using Nα1004760).

50% formamide, 5XSSC (composition: water 20 Om
In j, Na (J' 8.77g, Na5-citric acid 4
．． 41g) and a solution containing 0.1% SDS 6Tl11
Probe DNA (more than 30,000 CI) was added to the solution, treated at 100°C for 5 minutes, hybridized with the above-mentioned nitrocellulose filter at 42°C for 15 hours, and filtered with 2XSSC and 0.1% SDS solution. After washing, it was subjected to autoradiography.

As a result, the Pvul fragment (0.75 Kb) was found in the wild type N
In G13, it was detected at a position of 0.75 Kb, and in the recombinant strain R
16, it was detected at a position of approximately 1°05 Kb. This suggests that the synthetic promoter for constitutive expression of nifA (
0.3 Kb) was inserted into the PvuI fragment (see Figure 7).

In addition, the Sma I fragment is 3.3 Kb in wild strain NG13.
In the recombinant strain R16, it was detected at a position of 5.2 Kb, indicating that Tc' to nifL on the chromosome
This shows that insertion of a gene (1.6 Kb) and insertion of a synthetic promoter (0.3 Kb) upstream of nifA occurred (see Figure 8).

From these facts, recombinant strain R16 has ni on the chromosome.
The Tc' gene is inserted into the fL gene, resulting in nifL-
It was also confirmed that a synthetic promoter was inserted upstream of the nifA gene and that it had a nif AC marker.

Example 6 Measurement of nitrogen fixation activity based on acetylene reducing ability of recombinant strain R16: NG13 strain, R16 strain, a strain in which pNOA102 was introduced into R16 strain, and Pneumoniae UNF 7
No. 14 was cultured in 5+nl of L-broth at 37°C for 8 hours.

200 d of culture solution was added to a final concentration of 0.2% (NH4)2SO.

5 ml of nitrogen-free medium containing (MgSO40, 1 g.

NaMOOaj 2H2025mg, FasOn7Hz
025mg, glucose 20g, 2HP 0.12.
06g, KHzP○*3.4g, distilled water If, pH 7,
0), the cells were sealed with butyl rubber stoppers, and cultured at 30°C for 8 hours. Collect bacteria and add 5 ml of nitrogen-free medium or 0.2% (
The suspension was suspended in 5 ml of nitrogen-free medium containing NH4)2SOa, sealed with a butyl rubber stopper, and the gas phase was replaced with a mixed gas of 85% argon and 15% acetylene. The culture was incubated at 30°C with shaking (60 rpm) for 20 hours, and the produced ethylene was analyzed by gas chromatography (Shimadzugas Chr.
omatography GC-9A, Porapa
k T3Q~100p, 2128G column)
It was measured with

The results are shown in Table 1.

Table 1

[Brief explanation of the drawing]

FIG. 1 shows the structure and function of the nitrogen fixation gene group of K. pneumoniae. FIG. 2 shows the higher order regulation of nitrogen fixation gene expression in P. pneumoniae. Figure 3 shows the cloning of the entire nif gene group of K. oxytoca. FIG. 4 shows the construction steps of plasmid pNOY9Cm. FIG. 5 shows the construction steps of plasmid pNOA102. Figure 6 shows the introduction of plasmid pNOY9cm into the chromosome of K. oxytoca NG 13 and
The structure of the nlfLA portion of the chromosome of tocaR16 is shown. FIG. 7 shows the results of Southern hybridization, John, confirming that a synthetic promoter was inserted for constitutive expression of nifA on the chromosome of recombinant R16. In the figure, 1 is pBR328-Taql, 2 is NG13 chromosome-PvuI, 3 is pNOY8-Pvu, and 4 is R
Chromosome 16-PvuI. 5 indicates pNOY9cm-Pvu I. Figure 8 shows TC to nifL on the chromosome of recombinant R16.
The results of Southern hybridization involving the insertion of the r gene and the insertion of a synthetic promoter upstream of nifA are shown. In the figure, 1 is λ-3tyl, 2 is NG13 chromosome-3mal, 3 is pNOY8-3rna [4 is R1
Chromosome 6-3ma I, 5 is I) NOY9Cm-3m
a Indicates I. Patent applicant Tsukasa Uozumi (102) Kyowa Hakko Kogyo Co., Ltd.

Claims (6)

[Claims] (1) nifL gene deleted with marker, nifA
A recombinant plasmid containing the gene and a promoter integrated upstream of the nifA gene. (2) Recombinant plasmid pNOY9Cm. (3) A microorganism that belongs to the genus Klebsiella, has the ability to fix nitrogen, has the nifL gene on its chromosome deleted, has a promoter integrated upstream of the nifA gene, and has reduced inhibition of the ability to fix nitrogen in the presence of ammonia. (4) Klebsiella oxytoca R16. (5) The microorganism according to claim 3, further comprising a recombinant plasmid incorporating a constitutive promoter upstream of the nifA gene. (6) Klebsiella oxytoca R16 (pNOA10
2).