JP3169928B2 - Isolation of a Rhodobacter sphaeroides RV uptake hydrogenase (Hup) deficient strain with improved hydrogen light production efficiency - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mutant strain of Rhodobacter spheroides (Rh) which is unable to synthesize an uptake hydrogenase enzyme responsible for hydrogen consumption.
odobacter Sphaeroides) RV CBS 10080
5, and a method for producing hydrogen using the mutant strain.

[0002]

BACKGROUND OF THE INVENTION Hydrogen is the most abundant element in the sun and plays an important role in generating energy. However, hydrogen on earth, along with oxygen, is a fundamental compound for the biological energy cycle.

[0003] The amount of hydrogen contained in the lowest layer of the atmosphere can be calculated to be about 2.2 × 10 ⁹ tons equal to about per volume of air 0.55 ppm. Comparing this with the O ₂ content of 1.1 × 10 ¹⁵ tons, hydrogen is indeed considered a rare gas on earth. But,
Hydrogen, unlike noble gases, is constantly produced and used by a wide range of microorganisms, both in aerobic and anaerobic conditions, in the presence or absence of light.

[0004] From an energy point of view, hydrogen is an ideal source because it has a higher calorific value than anything else. In addition, hydrogen can be easily stored, is non-polluting,
The final oxidation product, water, is not only non-toxic,
It is an essential compound to guarantee life on earth.

[0005] The ability of microorganisms to generate hydrogen, along with the gradual depletion of traditional energy sources and the significant environmental problems associated therewith, has been tremendous for many years to obtain this energy source biologically. Has stimulated research efforts.

[0006] Many organisms capable of spontaneously producing hydrogen live in the natural environment.

Among these organisms, non-oxygen generating photosynthetic bacteria, such as purple non-sulfur bacteria (particularly Rhodopseudomonas sp., Rhodobacterium)
ter) and Rhodospirillum (including the genus Rhodospirillum) are particularly suitable for producing hydrogen. In fact, they combine the production of energy with the waste management, which is a significant environmental problem, because they can not only use light, a zero cost energy source, but also use waste material as a substrate for growth. Can be.

[0008] However, these bacteria have drawbacks derived from their low hydrogen production. As a result, the use of these microorganisms may result in the biological production of hydrogen (bi
oproduction) makes it difficult to develop methods.

[0009] A number of red non-sulfur bacteria, especially Rhodospirillum rubrum and Rhodobacter capsulata, are among others.
Atus), it is actually known in the literature that two different enzymes are involved in the metabolism of hydrogen. That is, a nitrogenase complex that is responsible for producing hydrogen, and a membrane hydrogenase called uptake hydrogenase that catalyzes the consumption of hydrogen.

[0010] The physiological role of the uptake hydrogenase is to oxidize the hydrogen produced by the nitrogenase, which is used as an energy source for cell growth, which results in a decrease in the rate and amount of hydrogen produced. Becomes

[0011]

SUMMARY OF THE INVENTION In order to improve the hydrogen-producing ability of Rhodobacter sphaeroides RV in terms of speed and efficiency, the present invention has now created a mutant strain that is unable to synthesize an uptake hydrogenase enzyme involved in hydrogen consumption.

This mutant, designated SMV089,
In accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure, on April 27, 1998, the deposit number C
In BS100805, the Central Bureau of Fungal Culture of the Netherlands (Cent
raalbureau Voor Schimmelcultures).

[0013] Accordingly, the present invention provides a mutant strain of Rhodobacter sphaeroides RV which cannot synthesize an uptake hydrogenase enzyme involved in hydrogen consumption.
CBS 100805.

It is a further object of the present invention that the mutant strain Rhodobacter sphaeroides RV CBS 100 is provided.
805 using 805.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [Ni-Fe] uptake hydrogenase is a heterodimeric enzyme consisting of a large subunit (hupL) and a small subunit (hupS).

Since these enzymes have been isolated from both aerobic and anaerobic bacteria and have a high degree of homology with respect to the primary sequence of their structural subunits, it is presumed that both bacteria are derived from a common ancestor. You.

Genetic analysis of incorporation-type hydrogenases isolated from various microorganisms reveals that the coding gene is composed of a plurality of functional units, and that their relative order is different, even if their relative order is different. It was shown that they were arranged regularly.

From a biochemical point of view, the large subunit has a catalytic function and the small subunit is involved in the transfer of electrons to specific carriers.

The present invention is based on the concept that in order to obtain a mutant strain in which hydrogen produced by nitrogenase cannot be reused, inactivating the large subunit results in greater damage at the structural level. Based on

Therefore, after genetically confirming the presence of this enzyme in the genome of Rhodobacter spheroides RV, which overproduces hydrogen, we proceeded with the gene encoding the large subunit of the uptake hydrogenase enzyme (hu
It was considered that the ability to consume hydrogen was eliminated by inactivating pL).

For this inactivation, conventional methods such as random mutagenesis based on the use of transposons or specific mutagenesis using interposons can be used. These components, after insertion into the genome, cause a disruption in the sequence of the gene (as revealed by phenotypic variation) and confer resistance to the particular antibiotic to the host.

According to a preferred embodiment of the present invention, a Rhodocobacter sphaeroides RV is inserted by inserting a genetic cassette (interposon) encoding resistance to the antibiotic Trimetoprim into a gene encoding the large subunit of an uptake hydrogenase enzyme. Of the wild-type synthase gene hupL, and a mutant strain was selected on a medium supplemented with the antibiotic.

In fact, theoretically, only clones in which the interposon has been integrated into the genomic DNA of the strain can grow on this selective medium.

The construction of the interposon involves isolating a fragment of about 2100 bp (SEQ ID NO: 1) corresponding to the 3 'region of the hupS gene and the 5' region of the hupL gene cloned from the wild-type strain Rhodobacter sphaeroides RV. It was done by

This fragment was cloned into the plasmid pUC18 (Boheringer) which had been previously modified to delete the PstI site from the multiple cloning sites.

Thereafter, plasmid p34E-T containing a gene encoding resistance to the antibiotic trimethoprim
p (D. De Shaezer and DEWoods (1996) BioTechniqui
es, 20: 762-764) was treated with the restriction enzyme PstI.

Next, a PstI-PstI fragment of about 650 bp containing a structural gene encoding resistance to trimethoprim and a promoter region upstream of the gene was ligated to a plasmid pUC18 containing a 2100 bp fragment isolated from the wild type of Rhodobacter sphaeroides. And the resulting ligase mixture was subjected to E. coli. col
i used to transform XL1-Blue cells.

The restriction analysis of the colonies obtained after the transformation confirmed the isolation of a positive clone containing the trimethoprim resistance gene (gene dhfrIIb encoding dihydrofolate reductase of E. coli) inserted into the gene hupL. Was. The nucleotide sequence of this clone revealed that the interposon was inserted in the reverse orientation with respect to the transcription direction of the gene encoding the large subunit of the uptake hydrogenase enzyme.

Subsequently, the disrupted gene hupL was cloned into a suicide vector, that is, a vector that could not self-replicate in Rhodobacter spheroides. The final construct is called E. coli. coli S17-1
The strain was introduced by conjugation with a wild-type strain of Rhodocobacter spheroides RV that was resistant to the antibiotic rifampicin.

The selection of mutant strains of Rhodobacter sphaeroides RV on trimethoprim allows
Successful recombination between the mutated copy of the gene hupL (carried by the suicide vector) and the functional copy on the genomic DNA was confirmed.

The clone was characterized by genetic amplification of the mutated fragment. In some cases, fragments of DNA were obtained in large sizes relative to the negative control, indicating the presence of the interposon internally.
Southern blot analysis was performed on this positive clone,
The nature and site of the mutagenesis were confirmed. The genomic DNA is extracted and the restriction enzymes MscI or Bst
XI and hybridized with a synthetic oligonucleotide specific for the gene hupL. Wild-type genomic DNA prepared under the same conditions was used as a negative control.

In this case, too, a hybridization band having the expected size was observed. This positive clone, designated SMV089, was characterized physiologically by growth on different media.

As with the wild-type strain, this clone was prepared in a complete medium (eg, aSy (J. Miyake, XY Mao and
S. Kawamura, J.A. Ferment.Technol., 62: 531-535,
1984)) and a specific medium for hydrogen production (E. Naka
da, Y. Kaji, K .; Aoyama, S.M. Nishikata, Y. Asad
a and J. Miyake) showed the same proliferation ability.

For clone SMV089, the determination of the activity of the uptake hydrogenase enzyme performed by spectrophotometric analysis proved to be negative, so that this mutant has no capacity to consume the produced hydrogen. It was confirmed that.

In addition, the activity of the nitrogenase responsible for the production of H ₂ was observed with surprising activity and was higher than that of the wild-type strain (46% higher on average).

Analysis of polyhydroxybutyrate (Bran
dl et al. (1988), Appl. Environ. Microbiol. 54, 1977
Β in the mutant strain
-The concentration of hydroxybutyric acid methyl ester was shown to be lower than that of the wild type.

Thus, the mutant strains of Rhodobacter sphaeroides RV of the present invention are particularly suitable for the development of fermentation processes for hydrogen production.

The fermentation can be carried out in an aqueous medium containing assimilable sources of carbon and nitrogen and various cations, anions and, optionally, trace amounts of vitamins or amino acids.

Sources of carbon assimilation include organic acids such as pyruvate, malate, lactate, succinic acid or other conventional carbon sources. The nitrogen source can be selected, for example, from inorganic ammonium salts (eg, ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium carbonate) and urea, or raw materials containing organic or inorganic nitrogen (eg, peptone, yeast extract or meat extract). .

Non-limiting examples of cations and anions can be selected from potassium, sodium, magnesium, iron, calcium, acid phosphates, sulfates, chlorides, manganese and nitrates.

According to a preferred embodiment of the process according to the invention, the raw material derived from the controlled acidogenic fermentation of municipal solid waste is a substrate having the composition shown in Example 9 (ED'Addario
(1992), Wat. Sci. Tech. 27, 183-192).

The fermentation is carried out under anaerobic conditions guaranteed by a continuous flow of argon, with light intensities ranging from 6,000 to 20,000 lux and temperatures ranging from 25 ° C. to 40 ° C., preferably Is performed at 30 ° C. to 37 ° C.

The pH of the fermentation medium is kept in the range from 6.50 to 8.0, preferably between 6.8 and 7.3. Adjustment of the pH can be performed, for example, by adding a basic aqueous solution such as an aqueous solution of ammonia, potassium hydroxide, sodium hydroxide, sodium carbonate or potassium carbonate. Preferably, a solution of 1-5 M sodium hydroxide is used.

Throughout the fermentation, hydrogen is recovered using conventional techniques.

The process can be performed continuously with an apparatus of the type illustrated in FIG.

[0046] When operating under the preferred conditions, production rate of H ₂ is in the mutant strain SMV089, it was observed to maintain a value higher than the rate of production of the wild-type strain throughout the entire experiment, the fermentation of the same period, the light-producing A total increase in hydrogen of 47% per liter of reactor and a 66% increase per milligram of dry weight of biomass was measured.

The excellent productivity observed in the mutant strain is associated with both the high activity of the nitrogenase enzyme and the inactivation of the uptake hydrogenase.

These results were confirmed in another experiment in which the mutants were fermented under favorable conditions using different lots of waste material as substrate. Despite the changes for different concentrations of components in different media lot, from the viewpoint of light production H _2, superior ability of the mutant lines was demonstrated once again by comparison.

The following examples are presented merely for purposes of illustrating the invention in more detail and should not be considered as limiting the scope of the invention.

[0050]

EXAMPLES Example 1: Rhodobacter spheroides R
Extraction of Genomic DNA from V 100 mL of LB medium (10 g / L tryptone, 5 g
/ L yeast extract, 10 g / L NaCl)
In a 0 mL flask, (National Institute of Bioscience and Human Engineering (NI
BH) (provided by Tsukuba, Japan) and inoculated with Rhodocobacter sphaeroides RV strain, and maintained the resulting mixture under aerobic / dark conditions at 30 ° C. for 48 hours with stirring (220 rpm). did.

Thereafter, SS34 rotor model Sorvall
Centrifuge the culture with RC-5B (5000 rpm at 4 ° C).
m, 10 minutes) and collect the cells.
Washed with E buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4) (2 × 120 mL). The resulting suspension is centrifuged again as above to collect the cells,
Resuspended in 9.5 mL TE buffer. 0.5 mL of 1
0% SDS (sodium dodecyl sulfate) and 50 μL
After addition of Proteinase K solution (20 mg / mL)
The suspension was incubated at 37 ° C. for 1 hour.

Next, a 0.7 M NaCl solution consisting of 1.8 mL of 5 M NaCl and 1.5 mL of 10% hexadecyltrimethylammonium bromide (CTAB) was added, and the resulting solution was incubated at 65 ° C. for 20 minutes.

The solution was then deproteinized with an equal volume of chloroform: isoamyl alcohol (24: 1, v / v) and the DNA precipitated with 0.6 volumes of isopropanol.

The DNA was washed with 1 mL of ethanol (70%) and collected using a glass rod. Finally, the recovered DNA was dissolved in 4 mL of TE buffer, and the concentration was adjusted to 26.
It was measured by a spectrophotometer reading at 0 nm.

The genomic DNA was purified again by centrifugation on a CsCl gradient containing 0.1 mg / mL ethidium bromide (Beckman V65Ti rotor for 16 hours at 55,000 rpm).

The DNA bands were visualized under UV light and the ethidium bromide was removed by extraction with butanol saturated in water. After dialysis against TE buffer, the DNA was precipitated with ethanol and resuspended to the desired volume.

Example 2: Isolation of a DNA fragment containing the 3 'end of the gene hupS and the 5' end of the gene hupL The description provided by Leung et al. (Leung DW, Chen E.
, Goeddel DV, Technique −a journal of methods
in cell and molecular biology, 1, Nr. 1 (198
9): The following pair of oligos synthesized according to the conserved nucleotide sequences of the genes hupS and hupL according to pages 11-15) and obtained by the IPCR (inverse PCR) technique using the genomic DNA of Rhodobacter sphaeroides DSM158. Genome D obtained as described in Example 1 using nucleotides as primers
NA was amplified by the polymerase chain reaction (PCR) technique.

(1) 5 'AATACAAGGG CAA
CTACATT C 3 '(forward) (2) 5' GCCCTCACC TGGTTCTTG
A TGTCCT 3 '(reverse) amplification was performed using a DNA thermal cycler 480 device (Perkin
-Elmer Cetus) using a reaction mixture (100 μL) containing:

500 ng of genomic DNA 10 mM Tris-HCl (pH 8.3) 2 mM MgSO ₄ 25 mM KCl 5 mM (NH ₄ ) ₂ SO ₄ 100 pmol of the two primers 200 μM dNTPs (dATP, dGTP, dCT
P, dTTP) 2.5 units of Pwo DNA polymerase (Boehri
The cycle program was started after denaturation at 98 ° C. for 2 minutes. This cycle program consists of a total of 25 cycles of 98 ° C. for 1 minute (denaturation) 60 ° C. for 1 minute (annealing) 72 ° C. for 2 minutes (extension), followed by 8 minutes at 72 ° C. (final extension). including.

The thus obtained amplification product is treated with phenol-chloroform (1: 1) to give NaCl
(1/10 vol / vol) and EtOH (2 vol
Precipitated in 1) and resuspended in 20 μL H ₂ O. Thereafter, a fragment of about 2100 bp was ligated to TAE (Tris acetate E
0.8% low melting gel in DTA) (SeaPlaque, FMC)
BioProducts). After elution from the gel, this fragment was converted to plasmid pUC18 (Bohe) which had been previously modified to remove the PstI site from the multiple cloning site.
ringer). In practice, this plasmid pUC18 is treated with the restriction enzymes HindII and SphI, flanking the PstI site in the polylinker; its ends are blunt-ended by incubating with the Klenow enzyme of DNA polymerase for 15 minutes at 30 ° C. Ligated again using T4 DNA ligase.

The plasmid thus obtained was treated again with the restriction enzyme SmaI, and ligated in the presence of a DNA fragment (5 μL / mL of DNA) in 30 μL of the reaction mixture. 2 μL of this mixture was electrocompetent. coli XL1-Blue cells (Dower WJ et al., Nucleicacid).
Research, 16, 6127-6145, 1988). This transformant was selected on a plate of LB agar medium containing 100 μg / mL ampicillin.

Plasmid D extracted from positive clone
According to the analysis of NA, about 21 corresponding to the expected size was obtained.
The presence of a 00 bp fragment was indicated. The plasmid contained in the positive clone was named pSM824 (FIG. 1).

Thereafter, the plasmid was sequenced using a DNA sequencer ABI373 (PerkinElmer). Due to its sequence, a 2100 bp fragment
It was confirmed that the gene corresponds to a DNA region containing about 650 base pairs (bp) of the gene hupS and about 1450 bp of the gene hupL.

Example 3: Inactivation of the gene hupL. It contains the gene (dhfrIIb) which codes for resistance to the antibiotic trimethoprim; plasmid p34E-Tp (D.Sh
aezer and DEWoods (1996) BioTechniques, 20:76
2-764) (5 μg) with 5 units of restriction enzyme Pst
It processed at 37 degreeC for 1 hour using I (Boehringer).
The treated mixture was loaded on a 0.6% low melting agarose gel (Seaplaque) and run at 70 volts for 4 hours.

Thereafter, a fragment PstI-Pst of about 650 bp containing a structural gene encoding resistance to trimethoprim and a promoter region upstream of this gene.
I was eluted from the gel.

The plasmid pSM824 (2 μg) was similarly treated with the enzyme PstI. Thereafter, the treated plasmid pSM824 and fragments PstI-PstI were
Ligated in 20 μL ligase buffer in the presence of 1 unit of T4 DNA ligase using a plasmid: fragment ratio of 1: 5. The ligase reaction was performed at 16 ° C. for about 16 hours.

Thereafter, the ligase mixture was added to E. coli. co
li XL1-blue was used to transform electrocompetent cells. Selection of transformants
1.5 mg / mL trimethoprim and 100 μg /
The test was performed on a plate of LB agar medium containing mL of ampicillin.

Analysis of positive clones indicated the presence of a clone in which the resistance gene to trimethoprim was inserted as expected into the gene hupL. Restriction analysis and nucleotide sequence analysis indicated that the gene dhfrII was inserted in the reverse orientation with respect to the gene hupL.

Example 4: To recover the entire fragment of DNA containing the two sites of the genes hupS and hupL and consisting of the region where the interposon is already present inside hupL, in the presence of the forward and reverse primers described above Gene amplification was performed on positive clones using Pwo DNA polymerase.

At the same time, the suicide plasmid pWKR102A
Was treated with the restriction enzyme HindIII and treated with Klenow polymerase at 25 ° C. for 30 minutes in the presence of dNTPs to form compatible ends.

Thereafter, the amplification product was mixed with the vector prepared as described above, and the mixture was incubated in a ligase buffer containing 5 units of T4 DNA ligase. The reaction was performed at 23 ° C. for 16 hours.

The ligase mixture was subjected to E. coli by electroporation. E. coli S17-1 were used to transform competent cells. Transformants were selected at 20 μg /
Performed on agar LB plates containing mL chloramphenicol and 1.5 mg / mL trimethoprim. From the positive clones, clones containing a plasmid (pSM825 (FIG. 2)) into which the above amplification product was correctly inserted were isolated.

The clone containing pSM825 was SMC5
08.

Example 5: E. coli and Rhodobacter sphaeroides RV E. coli SMC508 strain was inoculated into 5 mL of LB medium containing 20 μg / mL of chloramphenicol,
The cells were cultured overnight at 37 ° C.

The wild-type strain of Rhodobacter sphaeroides RV was rendered resistant to the antibiotic rifampicin (Rif) by selective pressure of the antibiotic up to a maximum of 150 μg / mL.

[0076] Rhodobacter spheroides RV (R
The an if ^R) strain, aS of 40mL having the following constituents
Y medium was inoculated and cultured at 30 ° C. in the dark under aerobic conditions for 2 days: (NH ₄ ) ₂ SO ₄ 1.25 g / L;
Succinic acid 9.8 g / L; yeast extract 1 g / L; K ₂
0.75 g / L of HPO ₄ ; 0.85 g of KH ₂ PO ₄
/ L; EDTA 2 mg / L; H ₃ BO ₃ 2.8 mg
_{/ L; Na 2 MoO 4 ·} 2H 2 O 0.75mg / L;
_{ZnSO 4 · 7H 2 O 0.24mg /} L; MnSO 4
・ 4H ₂ O 1.6mg / L; CuSO ₄・ 5H ₂ O
0.041 mg / L; CaCl ₂ .2H ₂ O 0.75
mg / L; MgSO ₄ .7H ₂ O 0.2 mg / L; F
_{eSO 4 · 7H 2 O 10mg /} L, pH7.0.

Then, Rhodobacter spheroides
3 mL of the RV (Rif ^R ) culture was mixed in a Falcon® tube with SMC508 in an amount corresponding to 1/10 of the optical density of the Rhodobacter sphaeroides RV culture.

The bacteria were centrifuged at room temperature for 10 minutes (30 minutes).
00 rpm) and resuspended in 100 μL of aSy medium, and a portion of this suspension was plated on the same agar plate.

When the plate was incubated at 35 ° C. for 6 hours, an exchange of genetic material between the two types of cells was observed. The cells were then harvested from the plate and
Resuspend in 00 μL aSy medium and add rifampicin (1
50 μg / mL) and trimethoprim (25 μg / m
L) on a plate of aSy medium containing

After 10 days of growth under aerobic conditions in the dark, a number of trimethoprim-resistant (Tp ^R ) and rifampicin-resistant (Rif ^R ) colonies were obtained on each plate.

[0081] Ten colonies were selected for detailed characterization. Genomic DNA was extracted from these clones and a PCR experiment was performed on the DNA product using the following oligonucleotide primers: (3) 5 'AACGTGGGACG ATCAGGGCCA
T CAT 3 '(forward) (4) 5' CCATAGGCCAT ACCAGGAGT
G ATCGA 3 '(reverse).

The expected amplification product is a fragment of the gene hupL, the presence of an interposon in this fragment
Revealed by an increase in size in the approximately 650 bp product (FIG. 3).

One of the 10 clones produced a fragment of the expected size (1600 bp), which results from a double crossover process on genomic DNA.
Another clone is a recombination by a single crossover that causes gene duplication on genomic DNA, ie, due to the presence of the mutant gene adjacent to the wild-type gene, two fragments (1
600 bp and 950 bp, corresponding to the wild-type fragment). The remaining eight clones produced wild-type fragments exclusively, confirming that they naturally had resistance to the antibiotic trimethoprim.

To confirm the site of mutagenesis, an experiment was performed by Southern blot analysis using genomic DNA isolated from only one positive clone.

The Rhodobacter sphaeroides RV wild-type (Rif ^R ) strain and its mutant were isolated at 20 μg / m 2.
Inoculate 5 mL of LB containing Kanamycin L
For 2 days in the dark under aerobic conditions.

Thereafter, genomic DNA was extracted and treated with restriction enzymes MscI or BstXI.

The processed product was analyzed on an agarose gel by electrophoresis. After separation by electrophoresis, Southern blot was performed according to a standard method, and the membrane on which the DNA was immobilized was hybridized with a probe specific to the gene hupL.

By autoradiographic development, B
In the genomic DNA of the mutant strain treated with stXI, it was shown that there was a single band of about 700 bp which was larger than that of the wild type strain (FIG. 4). The presence of this band confirmed that the gene hupL present on the genomic DNA was inactivated by interposon mutagenesis, since a double crossover occurred.

This mutant is indicated by the abbreviation SMV089.

Example 6 Growth of Mutant SMV089 Mutant SMV089 was cultured in various media and its growth was compared to that of a wild-type strain.

The medium used had the following composition: (A): (NH ₄ ) ₂ SO ₄ 1.25 g / L;
Succinic acid 9.8 g / L; yeast extract 1 g / L; K ₂
0.75 g / L of HPO ₄ ; 0.85 g of KH ₂ PO ₄
/ L; EDTA 2 mg / L; H ₃ BO ₃ 2.8 mg
_{/ L; Na 2 MoO 4 ·} 2H 2 O 0.75mg / L;
_{ZnSO 4 · 7H 2 O 0.24mg /} L; MnSO 4
・ 4H ₂ O 1.6mg / L; CuSO ₄・ 5H ₂ O
0.041 mg / L; CaCl ₂ .2H ₂ O 0.75
mg / L; MgSO ₄ .7H ₂ O 0.2 mg / L; Fe
_{SO 4 · 7H 2 O 10mg /} L, pH7.0.

(B): Na-lactic acid 98% 7.4m
L; Na L-glutamic acid 1.88 g / L; NaH
CO ₃ 1.5 g / L; K ₂ HPO ₄ 0.75 g /
L; KH ₂ PO ₄ 0.85 g / L; EDTA 2 mg
/ L; H ₃ BO ₃ 2.8 mg / L; Na ₂ MoO _{4.
2H 2 O 0.75mg / L; ZnSO} 4 · 7H 2 O
0.24 mg / L; MnSO ₄ .4H ₂ O 1.6 mg
/ L; CuSO ₄ .5H ₂ O 0.041 mg / L; Ca
_{Cl 2 · 2H 2 O 0.75mg /} L; MgSO 4 · 7
_{H 2 O 0.2mg / L; FeSO} 4 · 7H 2 O 10
mg / L.

The medium was sterilized by filtration using a 0.2 μl Nalgene filter, and all traces of oxygen were removed by bubbling argon for 30 minutes.

The culture solution was heated at 30 ° C. under a light intensity of 9,500 lux using a tungsten lamp.
For 3 days in an anaerobic test tube containing 24 mL of medium. At the end of the growth, the optical density is 60
It was measured at 0 nm. Table 1 shows the results.

[0095]

[Table 1]

From the values shown in Table 1, the mutation in the gene hupL indicates that the mutation in the synthetic medium (B) stimulates the production of hydrogen.
Even within, it can be observed that the growth ability of the mutant strain is not negatively affected.

Example 7: Hydrogenase uptake test To demonstrate the catalytic activity of the uptake hydrogenase enzyme, an in vitro colorimetric test was set up based on the decolorization of the methylene blue solution.

The hydrogen is oxidized in the presence of the active uptake hydrogenase enzyme, and the electrons are converted to an artificial acceptor (in our case,
(Methylene blue) which changes color when reduced.

The disappearance of the blue color resulting from the reduction of the artificial electron acceptor can be tracked at the appropriate wavelength with a spectrophotometer.

The test was carried out as follows: 1 ml of a solution of 20 mM Tris-HCl, pH 8 and 120 μM methylene blue was filled into a threaded glass cuvette equipped with a blind nipple with a pierceable diaphragm. The cuvette was sealed and a hydrogen molecule was bubbled through the solution for 5 minutes using a needle that pierces the rubber septum.
Thereafter, 50 μL of the anaerobic culture of the mutant strain (or Rhodobacter spheroides RV, used as a positive control) was injected with a microsyringe, the cuvette was immediately placed in the spectrophotometer and the decolorization kinetics of this solution Analysis was performed at 570 nm. The slope of the resulting curve is directly proportional to the activity of the uptake hydrogenase enzyme.

Example 8: Nitrogenase test To demonstrate the catalytic activity of the nitrogenase enzyme, an in vitro gas chromatography test was set up based on the reduction of acetylene. Acetylene is reduced to ethylene in the presence of active nitrogenase. This reaction is very specific because other enzyme systems cannot reduce acetylene. The loss of acetylene and the relative appearance of ethylene can be followed by gas chromatography analysis.

The test was performed as follows: 6 mL of culture was removed from the photobioreactor, maintained under an argon atmosphere, and transferred to a 20 mL tube equipped with a sealing plug. The samples were incubated at 30 ° C. for 5 minutes with stirring and under light supplied by a 200 watt lamp. Thereafter, 2 mL of acetylene was added, and 50 μL of head space was set for the set time (0, 5, 1).
5 and 20 minutes) and analyzed by GC. The column used was a HaySe with a length of 2 m and a diameter of 1 mm.
p (R) S, the analysis of which was performed at 45 ° C, 6 mL /
The analysis was performed with a He (carrier) flow of 5 minutes and an analysis time of 5 minutes (J. Meyer et al. (1978), J. of Bact. 136, 201-2).
08).

The results obtained show that the mutant SMV089
Shows that the nitrogenase activity is higher than that of the wild-type strain. This high activity can be estimated at about 46% or more (FIG. 7).

Example 9: Hydrogen photoproduction Hydrogen photoproduction for mutant SMV089 was determined in a fermentation experiment performed in parallel with the wild-type strain Rhodocobacter sphaeroides RV using the same lot of medium (Lot 1). In addition, two lots of media (Lot 1 and 2) were used to compare the results obtained for mutant SMV089.

The above mutant strain was subjected to a magnetic stirrer, a module for controlling pH, a 2100 watt halogen lamp installed inside to ensure illumination of the culture solution, and a temperature of 30 ° C. (See FIG. 5) in a 1 liter chemostat with a cooling system.

Pre-inoculations for fermentation were prepared in two sequential courses: 1.25 mL of glycerate in 1.25 mL of aSy medium at 30 ° C., aerobic / dark for 2 days. 1. Incubated; A 1:10 dilution of the previous culture was used for subsequent inoculation into anaerobic test tubes containing 60 mL of the same medium used for growth on the chemostat. The tubes were incubated at 30 ° C. under anaerobic conditions, under light illumination (tungsten lamp, 9,500 lux) for 2 days.

The second inoculum was diluted by calculating the initial optical density at 600 nm to 0.2 and inoculated into two chemostats.

The medium used is derived from a controlled acidogenic fermentation of municipal solid waste (ED'Addario et al. (19).
92), Wat. Sci. Tech. 27, 183-192). The average composition of this type of medium is as follows: total solids 3.7% volatile solids 72.0% lactic acid 36 g / L acetic acid 2.2 g / L total nitrogen 594 mg / L ammonia nitrogen 260 mg / L Total carbon 15.80 g / L TOC (total organic carbon) 14.90 g / L COD (chemical oxygen demand) 5.90 g / L Calcium 5.90 g / L Sodium 0.25 g / L Sulfate 0.25 g / L phosphate 23.00 mg / L For fermentation of the strain, the medium was first diluted 1:10 with distilled water to a lactic acid concentration of 3.6 g / L and then the following components were added: : 0.4 g of KH ₂ PO ₄ /
_{L; H 3 BO 3 2.8mg /} L; Na 2 MoO 4 · 2
_{H 2 O 0.75mg / L; ZnSO} 4 · 7H 2 O
0.24 mg / L; MnSO ₄ .4H ₂ O 2.1 mg
/ L; Cu (NO ₃ ) ₂ .3H ₂ O 0.04 mg / L;
CaCl ₂ .2H ₂ O 0.75 mg / L; MgSO ₄
・ 7H ₂ O 0.1mg / L; FeSO ₄・ 7H ₂ O
10 mg / L, pH 7.0.

The anaerobic conditions were maintained with a continuous argon flow. The light intensity used was adjusted to 6,000 lux and the two fermentations were started batchwise.

After about 24 hours, when the production of hydrogen began, the fermentation continued.

The flow rate (F) of the medium in the chemostat was 31
mL / h and the reactor volume (V) was 9
Since it was 30 mL, the equation D = F / V (D is the dilution rate)
According to this, the value of HRT (hydraulic residence time) (1 / D) was 30. That is, theoretically every 30 hours, the photobioreactor underwent a full turnover of the medium.

The fermentation was followed by evaluating the following analytical parameters: 2. Optical density measured at 600 nm to track bacterial growth; 2.1 pH automatically maintained at a value of 7.0 by adding NaOH; Total biogas produced. This was measured by a digital meter connected to one of the outlets of the chemostat; Percentage of hydrogen contained in biogas (FIG. 4). This is a Carbosieve II column 80-100 mesh
4. Measured on a VARIAN 3400 gas chromatograph equipped with a Supelco 200 x 3 mm; The amount of polyhydroxybutyrate accumulated by the cells (by gas chromatography analysis).

The light intensity was measured using an LSI lux meter before inoculation on the outer surface of the reactor.

From the value of biogas produced and the percentage of hydrogen present, the amount of hydrogen generated per day was determined per liter of reactor (mL H ₂ / L reactor / day) and per mg dry weight (mL H _Two
/ Mg dry weight / day).

The following table shows data from two separate experiments. Here, first, the mutant SMV08
9 was compared to that of wild type under the same fermentation conditions (medium 1 of the same lot). Thereafter, the ability of the mutant strain under different fermentation conditions (ie using two different media 1 and 2 in different lots) was confirmed.

In particular, Table 2 shows the hydrogen production values per liter of reactor calculated based on the values of total biogas measured by gas chromatography and the percentage of hydrogen. This value was found to be higher in the mutant SMV089 than in the wild type strain.

[0117]

[Table 2]

Table 3 shows the hydrogen production values per milligram dry weight calculated by the same procedure as described above.

[0119]

[Table 3]

The fermentation of the strain was stopped when the production of hydrogen reached insignificant levels. In terms of duration, the photoproduction of hydrogen in the mutant SMV089 is comparable to that obtained in the wild type strain. On the other hand, as far as the rate per day is concerned, the mutant strain remains higher than the wild type throughout the entire experiment, and the total amount of hydrogen photo-produced in the same fermentation time is 47% per liter of reactor. , And a 66% increase per milligram of dry weight.

The ability of the mutant strains to be evaluated starting from different lots of substrate also shows a high total amount of hydrogen produced and a variable range of gas values measured between 0.3% and 7%. Thus, excellent production capacity was established independent of the variability in the concentration of the various components in the media of the different lots.

Gas Chromatographic Analysis of Polyhydroxybutyrate (Brandl et al. (1988) Appl. Eviron. Microb
iol. 54, 1977-1982) showed the presence of peaks characteristic of β-hydroxybutyric acid methyl ester in both strains. PHB has reached a maximum accumulation of about 5% per dry weight on the last day of fermentation in both mutant and wild-type strains, but its concentration in the mutant strains has been increased over all experiments by wild-type It is lower than stocks.

[0123]

[Sequence List] SEQUENCE LISTING <110> Eni Technologie <120> Mutant of Rhodobacter Sphaeroides RV and Its Use in the Production of Hydrogen <130> B0098P0670 <140> <141> <150> IT / MI98A001224 <151> 1998-06- 03 <160> 5 <170> PatentIn Ver. 2.0 <210> 1 <211> 21 <212> DNA <213> Rhodobacter sphaeroides <400> 1 aatacaaggg caactacatt c 21 <210> 2 <211> 26 <212> DNA < 213> Rhodobacter sphaeroides <400> 2 gccctcgacc tggttcttga tgtcct 26 <210> 3 <211> 23 <212> DNA <213> Rhodobacter sphaeroides <400> 3 aacgtggacg atcagggcat cat 23 <210> 4 <211> 25 <212> DNA < 213> Rhodobacter sphaeroides <400> 4 ccataggcat accaggagtg atcga 25 <210> 5 <211> 2076 <212> DNA <213> Rhodobacter sphaeroides <400> 5 tacaagggca actacattct tgccgtcgag ggcaacccgc cgctgaacga ggacgggatg 60 tattgcatca tcggcggcaa gcccttcgtg gaacagctga agatggcggc cgaacatgcc 120 aaggccatca tcagctgggg ggcctgcgcc tcctacggct gcgtgcaggc ggcggcaccc 180 aacccgacgc gggcgacgcc ggtgcacaag gtcatcctcg acaagccgat catcaaggtg 240 ccgggctgcc cgcccatcgc cgaggtgatg acc ggcgtca tcacctacat gctgaccttc 300 gaccggctgc ccgagctcga ccgtcagggc cgcccggcga tgttctacag ccagcgcatt 360 cacgacaaat gctaccgccg cccgcatttc gacgcgggcc agttcgtcga ggcctgggac 420 gacgactacg ccaagaaggg ctactgcctc tacaagatgg gctgcaaggg gccgaccacc 480 tacaacgcct gctcgaccgt gcgctggaac gagggcgtaa gcttcccgat ccagtccggc 540 cacggctgca tcggctgctc ggaggacggc ttctgggatc aggggtcctt ctacgaccgg 600 ctgacgacga tcaagcagtt cggcgtcgag gccaatgccg acacgatcgg cctcacggcc 660 gtgggcgccc tcggcgcggg cgtggcggcg catatcgcgg ccaccgccct caagagcgcg 720 cagaagaaat cgcaggcggc gaatgccgcg aagaccgacg aaaagacgga ggcctgagcc 780 atggtcgcga caccgaacgg tttcaacctg gacaattcgg gccgccgcat cgtggtggac 840 cccgtgaccc ggatcgaggg tcacatgcgc tgcgaagtga acgtggacga tcagggcatc 900 atccgcaacg ccgtctcgac ggggaccatg tggcgggggc ttgaagtgat cctgaagggc 960 cgcgatccgc gcgacgcctg ggccttcacc gagcggatct ggggggtctg caccggcacc 1020 catgcgctca cctccgtccg cgcggtcgag gatgcgctgg ggatctcgat ccccgacaat 1080 gcgaactcga tccgcaacat gatgcagctg aacctgcaga tccacgacca cgtcgtccat 1140 ttctaccatc tgcatgcgct ggactgggtg aaccctgtca atgcgctgcg cgccgatccc 1200 aaggccacgt ccgagctgca gcagaaggtc tcgccctcgc atccgctctc gtcgccgggc 1260 tatttccgcg acgtgcagaa ccggctgaag aagttcgtgg aatcgggcca gctcggcctg 1320 ttcaagaacg gctactggga caatgcggcc tatctgctgc cgcccgaggc ggacctgatg 1380 gccacgacgc attacctcga ggcgctcgac ctccagaagg agatcgtcaa ggtccacacg 1440 atcttcggcg gcaagaaccc gcatccgaac tggctggtgg ggggcgtgcc ctgcccgatc 1500 aacatcgacg gcgtgggcgc ggtcggcgcg atcaacatgg agcggctgaa cctcgtctcc 1560 tcgatcatcg accagtgcat ccagttcacc aacaacgtct atctgcccga cgtggtggcc 1620 atcggcggct tctaccgcaa ctggctctat ggcggcgggc tctcgtcgaa gtcggtgatg 1680 gcctatggcg acatccccga gcatccgaac gatttctcgc ccggacagct gcacctgccg 1740 cgcggcgcga tcatcaacgg caatctcgag gaggtgcatg acgtcgaccc gcgccacccc 1800 gagcaggtgc aggaattcgt cgatcactcc tggtatgcct atggcgagcc ggggcgcggc 1860 ctgcacccct gggacggcgt gaccgagccg cgctacgagc tcggccccaa tgccaagggc 1920 acgcggacga acatcctcga gctcgacgag gcggcgaaat attcctggat caag gcgccg 1980 cgctggaagg ggcacgcgat ggaggtgggc ccgctcgccc gctacatcgt gggctatgcc 2040 aagggccacg aggacatcaa gaaccaggtc gagggc 2076

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a restriction map of plasmid pSM824.

FIG. 2 is a schematic diagram of a restriction map of plasmid pSM825.

FIG. 3 is a view showing the results of analysis on agarose gel of PCR products obtained using genomic DNAs of nine mutant strains as a template.

FIG. 4 shows the results of Southern blot analysis of genomic DNAs of mutant clone SMV089 and Rhodobacter sphaeroides RV strain and wild-type DSM treated with restriction enzymes MscI and BstXI and hybridized with a probe specific to gene hupL. FIG.

FIG. 5. Rhodocobacter sphaeroides R showing the presence and absence of the activity of the uptake hydrogenase enzyme (represented by the slope of the kinetic line), respectively.
The figure which shows the analysis result by the spectrophotometer of the wild type strain of V and the mutant strain SMV089.

FIG. 6 is an explanatory view showing a bioreactor.

FIG. 7 shows the trends in nitrogenase activity (expressed as rate of substrate disappearance per milligram dry weight) in wild-type and mutant SMV089 during hydrogen photoproduction experiments.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Substrate tank 2 ... Argon gas which flows to maintain anaerobic conditions 3 ... Peristaltic pump which adjusts the entrance of a photobioreactor 4 ... Photobioreactor 5 ... Magnetic plate to keep stirring the inside of a photoreactor 6 ... Photo Electrical connection for halogen lamps installed inside the bioreactor 7 ... Common outlet for spent medium, biomass and biogas 8 ... Trap 9 ... Digital meter of generated biogas 10 ... Emission of biogas 11 ... Peristaltic pump for discharging spent medium and biomass 12 ... Discharge and sanitation of medium and biomass

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/21 C12R 1:01) (C12N 15/09 ZNA C12R 1:01) (C12P 3/00 C12R 1:01) ( 72) Inventor Elisabetta Franchi 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building 8th Floor Global Environment Institute of Industrial Science and Technology (CO2) Project Room, etc. (72) Inventor Claudio Toge Tokyo 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Toyo Maritime Building 8th Floor, Institute for Global Environment, National Institute of Advanced Industrial Science and Technology (CO2) Project Room (72) Inventor Giuseppe Scolla 2--8 Nishi-Shimbashi, Minato-ku, Tokyo No.11 7th Oriental Maritime Building 8th Floor Global Environment Institute of Advanced Industrial Science and Technology Project Chamber (72) Inventor Francesco Rodriguez 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building 8th Floor Global Environment Institute of Industrial Science and Technology CO2 Fixed Project Room (72) Inventor Paola・ Pedroni 7-8 Toyo Maritime Building 8th Floor, 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo Project for the Interior of the Project for Fixing CO2, etc. (72) Gino Della Penna, Minato-ku, Tokyo 2-8-11 Nishishinbashi 8th floor of the 7th Oriental Maritime Building The Institute for Global Environment, National Institute of Advanced Industrial Science and Technology (CO2) Project Room (56) References Appl. Microbiol. Biotechnol. , 40 (5), 687-690 (1994) Appl. Microbiol. Biotechnol. , 37 (4), 496-500 (1992) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/20-1/21 BIOSIS (DIALOG) WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A mutant strain of Rhodobacter spheroides RV, which is not capable of expressing incorporation-type hydrogenase activity and has been assigned the deposit number CBS 100805. 2. The mutant strain of Rhodobacter spheroides RV according to claim 1, which expresses a higher nitrogenase activity than the wild-type strain. 3. A method for photoproducing hydrogen comprising: a) a mutant Rhodocobacter sphaeroides RV.
CBS 100805 was prepared in an aqueous medium containing assimilable carbon and nitrogen sources and various cations and anions under anaerobic conditions at a temperature range of 25 ° C to 40 ° C, 6.5 to 8.0 ° C. pH range, 6,000 to 2
A method comprising culturing under a light intensity range of 000 lux, and b) separating and recovering the hydrogen produced. 4. The method of claim 3, wherein said assimilable carbon source is selected from organic acids consisting of pyruvate, malate, lactate, and succinate. 5. The nitrogen source is selected from inorganic ammonium salts consisting of ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium carbonate, urea, and a raw material containing organic nitrogen or inorganic nitrogen consisting of peptone, yeast extract and meat extract. Item 3. The method according to Item 3. 6. The method of claim 3, wherein said cations and anions are selected from potassium, sodium, magnesium, iron, calcium, acid phosphates, sulfates, chlorides, manganese and nitrates. 7. The method of claim 3, wherein said fermentation medium is a feedstock derived from controlled acidogenic fermentation of municipal solid waste. 8. The method according to claim 3, wherein said aqueous medium contains vitamins or amino acids. 9. The method of claim 3, wherein said pH is selected from between 6.8 and 7.3. 10. The method of claim 3, wherein said temperature is selected from between 30 ° C. and 37 ° C. 11. The method of claim 3, wherein the method is performed continuously.