WO2015125849A1 - PROCÉDÉ DE PRODUCTION EN MASSE DE BACTÉRIOCHLOROPHYLLE b, ET SOUCHE DE PRODUCTION - Google Patents

PROCÉDÉ DE PRODUCTION EN MASSE DE BACTÉRIOCHLOROPHYLLE b, ET SOUCHE DE PRODUCTION Download PDF

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WO2015125849A1
WO2015125849A1 PCT/JP2015/054552 JP2015054552W WO2015125849A1 WO 2015125849 A1 WO2015125849 A1 WO 2015125849A1 JP 2015054552 W JP2015054552 W JP 2015054552W WO 2015125849 A1 WO2015125849 A1 WO 2015125849A1
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gene
producing
bchz
bchl
bchy
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祐介 塚谷
均 民秋
二朗 原田
祐一 藤田
次郎 野亦
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学校法人立命館
学校法人 久留米大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system

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  • the present invention relates to a recombinant bacteriochlorophyll b high-producing bacterium, a method for producing the same, and a method for mass production of bacteriochlorophyll b using the high-producing bacterium.
  • Bacteriochlorophyll is a name given to tetrapyrroles found in non-oxygen-producing photosynthetic bacteria. There are 7 types from a to g, and 6 types except BChl f so far. It is found from nature. Among these, BChl a, BChl b and BChl g have a bacteriochlorin skeleton in which the CC double bond at the ⁇ -position of both the B and D rings of the tetrapyrrole ring is reduced to a single bond.
  • BChl a and BChl b are present in red photosynthetic bacteria and BChl g are present in heliobacteria, and form non-covalent bonds with oligopeptides to form dye-protein complexes.
  • the absorption spectrum of (bacterio) chlorophyll has four absorption bands from the long wavelength side: Qy, Qx, By, and Bx (By and Bx bands are collectively referred to as the Soray band), but BChl a / has a bacteriochlorin skeleton.
  • the Qy band is greatly shifted in the near infrared region by a longer wavelength than BChl c / d / e and chlorophyll having a chlorin skeleton.
  • BChl b has a longer wavelength shift in the Qy band than BChl a due to the presence of the ethylidene side chain at the C8 position. Therefore, BChl b, bacteriochlorophyllide b (BChlide b) from which the long-chain hydrocarbon group in the propionate residue on the 17th position is removed, bacteriopheophytin b (BPhe b) and bacteriopheophore from which the central metal is eliminated. If bide b (BPheoide b) can be supplied at low cost and in large quantities, it is useful for the production of optical devices with improved light energy conversion efficiency.
  • BChlide b bacteriochlorophyllide b
  • BPhe b bacteriopheophytin b
  • bacteriopheophore from which the central metal is eliminated.
  • 8-vinyl chlorophyllide a (8V-Chlide a) is reduced to ethyl group by the action of 8-vinyl reductase (or divinyl reductase, called DVR).
  • DVR divinyl reductase
  • a (Chlide a) is generated.
  • chlorophyllide a oxidoreductase (COR) consisting of three proteins, BchX, BchY and BchZ, turns the double bond at the C7-8 position of the B ring into a single bond.
  • Bacteriochlorophyllide a (3V-BChlide a) is produced (Non-patent Document 1), and the C3 position is acetylated by the action of 3-vinyl bacteriochlorophyllide hydratase (BchF) and 3-hydroxyethylbacteriochlorophyllide dehydrogenase (BchC). After being converted to BChlide a, a long-chain hydrocarbon group is added to the propionate residue at position 17 by the action of BchG to generate BChl a.
  • BchF 3-vinyl bacteriochlorophyllide hydratase
  • BchC 3-hydroxyethylbacteriochlorophyllide dehydrogenase
  • BchX / Y / Z reduces the conjugated diene of the B ring of the chlorin ring of 8V-Chlide a by 1,4 addition and reduces the bacterio BChlide g, which is a precursor of BChl b and BChl g, is produced directly from 8V-Chlide a by catalyzing the reaction of providing an ethylidene group at the C8 position of the chlorin ring (Patent Document 1).
  • BChF and BchC act to acetylate the C3 position of BChlide g to produce BChlide b, and then BchG acts to produce BChl b, as in BChla-producing bacteria.
  • BchF / BchC does not exist in BChl g-producing bacteria, and BChl g is produced from BChlide g by the action of BchG.
  • an object of the present invention is to provide a BChl b high-producing bacterium, and to provide a method for mass-producing BChl b using the bacterium.
  • the inventors of the present invention have proposed that the reaction from BChlide g to BChl b in BChl b-producing bacteria is the same as the reaction from 3V-BChlide a to BChla in BChla-producing bacteria, as well as the acetyl group of the C3-vinyl group by BchF and BchC.
  • BchX / Y / Z (hereinafter referred to as “BvCOR”) derived from BChl b producing bacterium Blastochloris viridis and Rhodobacter sphaeroides It was tried whether BChlb could be produced by expressing. However, it alone did not lead to production of BChlb (FIG. 2). Several hypotheses were conceivable as the reason, but the present inventors originally found that R. spheroides was originally endogenous to R.
  • the bacterium has a third enzyme that can reduce 8V-Chlide a to Chlide a. It has been suggested (Canniffe, DP et al., Biochem. J., 450: 397-405 (2013)) that the present inventors confer the ability to produce BChlb to R. sphaeroides.
  • RcCOR BchX / Y / Z
  • BChl b could not be produced simply by expressing BvCOR in a wild strain of R. spheroides is that BchF and BchC endogenous to R. spheroides are not able to produce BChlide g or 3-hydroxyethylbacteriochlorophyllide b.
  • R. sphaeroides 2.4.1 and BChl b producing bacterium, Thioflaviococcus mobilis whose genome sequence is already known.
  • BchF and BchC show 71% / 80% and 56% / 69% amino acid identity / similarity respectively, but 80% / 90%, 70% / 75% and 65% / 77% respectively.
  • the present invention provides the following.
  • a photosynthetic bacterium that produces bacteriochlorophyll a it lacks the endogenous bciA gene and bchY and / or bchZ gene, and expresses bchY and bchZ genes derived from a photosynthetic bacterium that produces bacteriochlorophyll b or bacteriochlorophyll g.
  • Bacteriochlorophyll b producing mutants engineered as follows.
  • BChl b or a derivative thereof in which the Qy band is in the near-infrared region having a longer wavelength can be provided easily and in large quantities, and thus functional dyes having a changed light absorption band are used industrially. It becomes possible to do.
  • the endogenous bciA gene and bchY and / or bchZ gene are deleted, and bchY and bchZ genes derived from photosynthetic bacteria producing BChl b or BChl g are expressed.
  • An engineered BChlb producing mutant is provided.
  • the strain used as a parent strain for genetic manipulation is a photosynthetic bacterium capable of producing BChla, which grows quickly, can grow under dark (slight) aerobic conditions, and can be genetically manipulated. There is no particular limitation as long as it is correct.
  • Examples of BChl a-producing bacteria include R. spheroides, R.
  • capslatas Rhodospirillum photometricum, Rhodopseudomonas palustris, Thiocapsa marina, Thiocapsa marina, Allochromatium chromium vinosum) and the like, but is not limited thereto.
  • BChla-producing bacteria include, for example, ATCC, CAUP, CCAP, CCMP, CCCM, CGCCCC, CSIRO, DSMZ, Kagawa Prefectural Fisheries Experiment Station, Akashi Research Institute, Kobe University Seaweed Collection (KU-MACC), National Institute for Environmental Studies It can be obtained from culture collections of photosynthetic organisms, such as the Microbiology Preservation Facility (NIES) and the Biotechnology Center (NBRC) of the National Institute of Technology and Evaluation (NITE). For example, R. spheroides (NBRC 12203, 100037 and 100038) and R.
  • BChla-producing bacteria that can grow under microaerobic conditions and can be genetically modified are preferably R. spheroides, R. capsulatus, Rps. Pulstris, etc., more preferably R. spheroides and R. capsulatus, particularly preferably R. Spheroides is mentioned. If BChl a-producing bacteria, such as R. spheroides and R.
  • capsulatus have not established an efficient natural transformation / electroporation system, and if gene transfer is performed by conjugative transfer via E. coli, donor / In order to facilitate removal of the helper E. coli, it is preferable to use a rifampicin-resistant BChla-producing bacterium.
  • Rifampicin resistant bacteria can be easily obtained by, for example, seeding BChla-producing bacteria on a solid medium containing 100 ⁇ g / ml rifampicin and picking up naturally occurring resistant colonies.
  • the “bciA gene” to be deleted in the present invention is a gene having a coding region (cds) consisting of the nucleotide sequence shown in SEQ ID NO: 1 isolated from the R. sphaeroides genome (the amino acid encoded by the cds) The sequence is shown in SEQ ID NO: 2), as well as its orthologs in other BChla producing bacteria.
  • cds coding region consisting of the nucleotide sequence shown in SEQ ID NO: 1 isolated from the R. sphaeroides genome (the amino acid encoded by the cds) The sequence is shown in SEQ ID NO: 2), as well as its orthologs in other BChla producing bacteria.
  • capsulatus SB1003 strain whose entire genome sequences have been decoded are listed in the NCBI database as Gene ID: 552536492 (positions 111258-112292 (complementary strand) of NC_007494.2); locus tag : RSP_3070), and Gene ID: 294675557 (NC_014034.1, positions 3460325-3461290 (complementary strand); locus tag: RCAP_rcc03260), and those skilled in the art can easily obtain the genome sequence information of the gene and its upstream and downstream regions. Can be obtained.
  • the genome sequence is decoded by a conventional method, and then the Blast search is performed on the obtained genome sequence using the nucleotide sequence shown in SEQ ID NO: 1 as a query. Can be identified.
  • Confirmation that the hit gene is the bciA gene is, for example, whether 8V-Chlide a is brought into contact with a recombinant protein obtained by introducing an expression vector containing the gene into Escherichia coli to produce Chlide a. Can be done by examining. Based on the nucleotide sequence information of the bciA gene thus obtained, an appropriate oligonucleotide was synthesized as a primer, and a genomic DNA fraction prepared from a BChla-producing bacterium was used as a template. The target bciA gene can be amplified by “PCR method”. The nucleotide sequence of the DNA obtained as described above can be determined using a known sequence technique such as the Maxam-Gilbert method or the dideoxy termination method.
  • the “bchY gene” to be deleted in the present invention is a gene having a coding region (cds) consisting of the nucleotide sequence shown in SEQ ID NO: 3 isolated from the R. sphaeroides genome (the amino acid encoded by the cds) The sequence is shown in SEQ ID NO: 4), as well as its orthologs in other BChla producing bacteria.
  • cds coding region
  • capsulatus SB1003 strain whose entire genome sequence has been decoded are respectively listed in the NCBI database as Gene ID: 552535527 (1984719-1986227 positions of NC_007493.2 (complementary strand); locus tag : RSP_0261), and Gene ID: 294675557 (No. 759425-760918 in NC_014034.1; locus tag: RCAP_rcc00688).
  • Gene ID: 552535527 (1984719-1986227 positions of NC_007493.2 (complementary strand); locus tag : RSP_0261), and Gene ID: 294675557 (No. 759425-760918 in NC_014034.1; locus tag: RCAP_rcc00688).
  • Those skilled in the art can easily obtain the genome sequence information of the gene and its upstream and downstream regions. Can do.
  • the genome sequence is decoded by a conventional method, and then the Blast search is performed on the obtained genome sequence using the nucleotide sequence shown in SEQ ID NO: 3 as a query. Can be identified.
  • Confirmation that the hit gene is the bchY gene can be performed, for example, by examining whether or not the BChla production ability is lost when the gene is deleted.
  • an appropriate oligonucleotide was synthesized as a primer, and a genomic DNA fraction prepared from BChla-producing bacteria was used as a template, and the target bchY was obtained by PCR.
  • Genes can be amplified.
  • the nucleotide sequence of the DNA obtained as described above can be determined using a known sequence technique such as the Maxam-Gilbert method or the dideoxy termination method.
  • the “bchZ gene” to be deleted in the present invention is a gene having a coding region (cds) consisting of the nucleotide sequence shown in SEQ ID NO: 5 isolated from the R. sphaeroides genome (the amino acid encoded by the cds) The sequence is shown in SEQ ID NO: 6), as well as its orthologs in other BChla producing bacteria.
  • capsulatus SB1003 strain whose entire genome sequences have been decoded are listed in the NCBI database as Gene ID: 552535527 (NC_007493.2 at positions 1983244-1984719 (complementary strand); locus tag : RSP_0260) and Gene ID: 294675557 (positions 760918-762390 of NC_014034.1; locus tag: RCAP_rcc00689), and those skilled in the art can easily obtain the genome sequence information of the gene and its upstream and downstream regions. Can do.
  • the genome sequence is decoded by a conventional method, and then the Blast search is performed on the obtained genome sequence using the nucleotide sequence shown in SEQ ID NO: 5 as a query. Can be identified.
  • Confirmation that the hit gene is the bchZ gene can be performed, for example, by examining whether or not the BChla production ability is lost when the gene is deleted.
  • an appropriate oligonucleotide was synthesized as a primer, and a genomic DNA fraction prepared from a BChla-producing bacterium was used as a template.
  • Genes can be amplified. In many photosynthetic bacteria, the bchY gene and the bchZ gene are close to each other and partially overlap. In that case, both genes (may be referred to as bchYZ gene) may be cloned together. .
  • the nucleotide sequence of the DNA obtained as described above can be determined using a known sequence technique such as the Maxam-Gilbert method or the dideoxy termination method.
  • “Deleting an endogenous gene” in a BChl a-producing bacterium means that complete mRNA cannot be produced by destroying or removing the gene.
  • a target gene (genomic DNA) ⁇ derived from a target BChl a-producing bacterium is isolated according to a conventional method.For example, (1) ⁇ ⁇ ⁇ its coding region (cds) or other DNA in the promoter region Insert a fragment (e.g., drug resistance gene or reporter gene) cd to destroy the function of cds or promoter, or (2) cut out all or part of the target gene and delete the gene (e.g., drug (3) Insert a stop codon in cds to disable complete protein translation, or (4) DNA that terminates gene transcription into the transcription region By inserting a sequence (terminator sequence) ⁇ ⁇ ⁇ and disabling the synthesis of complete mRNA, the resulting DNA strand ⁇ (hereinafter referred to as a terminator
  • Tsu the computing vector and abbreviated may a scheme of incorporated target locus of interest BChl a producing bacteria is used by homologous recombination.
  • a drug resistance gene is inserted into cds to destroy the target gene, or all or part of the target gene is replaced with a drug resistance gene.
  • kanamycin resistance gene As the drug resistance gene, kanamycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, gentamicin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene and the like can be used.
  • the kanamycin resistance gene the neo gene contained in pUCKM1 (J. Biol. Chem., 266 (20): 12889-12895 (1991)) is used as the streptomycin and spectinomycin resistance gene, pHP45 ⁇ (Gene, 29: 303-313 (1984)), pSRA2, pSRA81 Method (Methods Mol.
  • the aadA gene is pMS255, pMS266 (Gene, 162: 37 -39 (1995)), the aacC1 gene contained in, erythromycin resistance gene and chloramphenicol resistance gene, ermC gene and cat gene contained in pRL409 (Gene, 68: 119-138 (1988)) It is done.
  • These genes can be excised from the plasmid with an appropriate restriction enzyme, or can be amplified by PCR using the plasmid as a template and the upstream and downstream regions of these genes as primers.
  • a targeting vector in which a drug resistance gene is inserted into cds of a target gene or all or part of the target gene is replaced with a drug resistance gene, see, for example, MethodsMethodin Enzymology, 497: 519-538 (2011) However, the method is not limited thereto, and a method well known in the technical field can be used as appropriate.
  • the vector By introducing the above targeting vector into the target BChl a-producing bacterium, the vector can be incorporated into the target locus of the target BChl a-producing bacterium by homologous recombination.
  • the gene transfer method is not particularly limited as long as it is a method applicable to the target BChl a-producing bacterium, and any method known per se (for example, natural transformation method, electroporation method, etc.) may be used.
  • any method known per se for example, natural transformation method, electroporation method, etc.
  • selection of homologous recombinants using gene transfer by conjugation transfer and drug selection using a suicide vector is a preferred embodiment. Methods in Enzymology, 497: 519-538 (2011)).
  • bciA gene isolated as described above or bchY and / or bchZ gene (hereinafter sometimes abbreviated as bchY / Z gene)
  • the gene is treated in cds.
  • a sequence homologous to the target enzyme gene at both ends of the inactivated enzyme gene by cutting or excluding a part of the gene and inserting the drug resistance gene isolated as described above. Construct a cassette containing Different drug resistance genes are inserted into the bciA gene and the bchY / Z gene, respectively.
  • the obtained cassette is obtained by using an appropriate suicide gene (for example, sacB gene derived from Bacillus subtilis (producing a fructose polymer that inhibits the growth of Gram-negative bacteria)) and the origin of replication of the donor E. coli (eg, colE1 Etc.) but is inserted into a vector (suicide vector) that cannot be replicated by the recipients R. spheroides or R. capsulatus to construct a targeting vector. Subsequently, donor E. coli is transformed with the targeting vector by a conventional method.
  • an appropriate suicide gene for example, sacB gene derived from Bacillus subtilis (producing a fructose polymer that inhibits the growth of Gram-negative bacteria)
  • a vector siicide vector
  • Transformation of R. sphaeroides and R. Capsulatus is performed by conjugating with E. coli (donor E. coli containing the targeting vector, helper E. coli that induces conjugation, and recipients such as R. Spheroides and R. Capsulatus. Or a parental transformant (donor E. coli) obtained by introducing the target expression vector into Escherichia coli having the ability to transfer conjugation with R. Spheroides or R. Capsulatus.
  • the donor Escherichia coli in the triple parental conjugation there are no particular limitations on the donor Escherichia coli in the triple parental conjugation, and a normal strain such as JM105 may be used, such as XL1-Blue, DH5, and C600 having the helper plasmid pDPT51.
  • Escherichia coli having conjugation transmission ability used for double parental joining include, (Bio / Technology, 1: 784-791 (1983)) The conjugation of donor Escherichia coli with the recipients R. Spheroides and R. Capsulatus (additional E. coli in the case of triple parental joining) is known per se.
  • rifampicin resistance for example, it can be easily done by selecting on rifampicin-containing medium. Contamination of donor and helper E. coli can be prevented.
  • a transformant can be obtained by culturing a bacterial cell into which a targeting vector has been introduced on a solid medium containing a drug and selecting resistant colonies.
  • the medium used here any medium suitable for the growth of the target BChla-producing bacterium can be used.
  • a medium recommended by a culture collection that stores BChla-producing bacteria can be used.
  • the BChla-producing bacterium is R. spheroides or R. capsulatus
  • medium No. 802 available from Wako Pure Chemical Industries listed in the medium information of online catalog search provided by NBRC Examples include Medium No. 360 (SA medium).
  • PYS medium 1% polypeptone, 0.5% yeast extract, 1% sodium chloride; J. Mol.
  • Evol., 45 (2): 131-136 (1997)) is also preferably used.
  • concentration of the selective agent added to the medium varies depending on the type of antibiotic and the type of strain.For example, in the case of R. sphaeroides 2.4.1, 25 ⁇ g / mL kanamycin, 10 ⁇ g / mL gentamicin, 50 ⁇ g, respectively. / mL Streptomycin, 50 ⁇ g / mL spectinomycin can be added to the medium to a final concentration. It is desirable to start the drug selection by plating the cells on a selective medium after culturing in a non-selective medium for about 1 to 3 days from the gene transfer treatment.
  • Cultivation is usually carried out at a temperature of 20-40 ° C., preferably 25-30 ° C., in a dark (slight) aerobic condition.
  • the targeting vector When the targeting vector is inserted into the target enzyme gene on the genome by one homologous recombination, it becomes drug resistant by the expression of the drug resistance gene and can grow on the drug-containing medium.
  • the suicide gene since the suicide gene is also inserted into the genome, for example, when the sacB gene is used, this insertion mutant cannot grow in the presence of 10% sucrose.
  • the strain can grow in the presence of drugs (antibiotics) and sucrose.
  • resistant colonies are obtained in about 1-2 weeks from the start of selection. It can be confirmed by genomic PCR and / or sequence analysis of the target gene region that the obtained resistant clone is a homologous recombinant (bciA gene (bchY / Z gene) -deficient mutant).
  • bciA gene bchY / Z gene
  • homologous recombination mutants lacking these genes can be obtained.
  • the obtained mutant strain can be cryopreserved ( ⁇ 70 or 80 ° C. or lower) in a medium containing 5% DMSO or 15% glycerol.
  • GTA Gene disruption method using phage-like particles
  • G. overcapillus eg, R121, Y262, CB1127
  • a targeting vector eg, R121, Y262, CB1127
  • a large amount of GTA accumulates in the medium.
  • Some of these have cut out targeting vectors and incorporated them into the particles. Therefore, when this culture filtrate is brought into contact with the recipient's R. Capsulatus, the targeting vector is introduced from the GTA into the recipient cell, and homologous recombination is induced, so that the target gene is efficiently targeted to the target locus. be able to.
  • BChl a-producing bacteria The introduction of bchY and bchZ genes derived from BChl b or BChl g-producing bacteria into BChl a-producing bacteria can be performed as follows.
  • the photosynthetic bacterium from which the bchY and bchZ genes introduced into BChl a-producing bacteria are derived is not particularly limited as long as it produces BChl b or BChl g.
  • BChl b-producing bacteria include Blastochloris viridis, Thioflavicoccus mobilis, Thiococcus pfennigii, Thioalkalicoccus limnaeus, Thioalkalicoccus ⁇ ⁇ limnaeus, Phobilis (Blastochloris sulfoviridis), Halorhospira halochloris (Halorhodospira halochloris) and the like, and BChl g producing bacteria, for example, Heliobacterium modesticaldum, Heliobacterium chlorum (Heliobacterium chlorum), Examples include Heliobacterium gestii, Heliophilum fasciatum, and Heliobacillus mobilis.
  • B. viridis T. mobilis
  • Cloning of enzyme genes is usually performed by the following method.
  • the enzyme is completely or partially purified from a cell or tissue that produces the desired enzyme, and its N-terminal amino acid sequence is determined using the Edman method or mass spectrometry.
  • the amino acid sequence of an oligopeptide obtained by partially decomposing the enzyme with a protease or chemical substance that cleaves the peptide in a sequence-specific manner is similarly determined by Edman method or mass spectrometry.
  • An oligonucleotide having a nucleotide sequence corresponding to the determined partial amino acid sequence is synthesized, and this is used as a probe from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme.
  • the DNA encoding the enzyme is cloned by a hybridization method.
  • an antibody against the enzyme is prepared according to a conventional method using all or part of the completely or partially purified enzyme as an antigen, and the antibody is prepared from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme.
  • DNA encoding the enzyme can also be cloned by a screening method.
  • the gene of an enzyme similar in enzymatic properties to the target enzyme is known, for example, the NCBI BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/) is accessed and the known gene Search for a sequence homologous to the nucleotide sequence of the gene, create a probe as described above based on the hit nucleotide sequence, and clone the DNA encoding the enzyme by colony (or plaque) hybridization be able to.
  • B. viridis-derived BchY and BchZ consisting of the amino acid sequences shown in SEQ ID NOs: 8 and 10, respectively, 33% respectively at the amino acid level with B. ch.
  • BchZ (SEQ ID NOs: 12 and 14) derived from H. And 37% identity (similarities are 53% and 54%, respectively), so that a homology search with the nucleotide sequences of BchY and bchZ genes from B. viridis (SEQ ID NOs: 7 and 9) revealed that H. ORFs (SEQ ID NOs: 11 and 13) encoding BchY and BchZ on the genome of Rudeum can be found.
  • RNA or mRNA fraction was used as a template for Polymerase Chain Reaction ( Hereinafter, it can also be directly amplified by “PCR method” or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
  • PCR method or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
  • RT-PCR method Reverse Transcriptase-PCR
  • the cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired.
  • the DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • Examples of large-scale expression vectors in R. Spheroides and R. Capsulatus include pJN3 and pJNY constructed from the broad host range vector pBBR1-MCS2 (Gene, 166: 175 (1995)) (Cryogenic Science, 67: 649- 653 (2009)). Both vectors control the expression of pucAB encoding a light-harvesting bacteriochlorophyll-binding protein, and have a cloning site downstream of the puc promoter that exhibits high transcriptional activity under anaerobic conditions, allowing insertion of DNA encoding the target protein. .
  • a 6xHN tag is inserted in pJN3 and a Strep tag is inserted in pJNY, facilitating purification of the expressed protein.
  • a streptomycin / spectinomycin resistance gene has been newly introduced as a drug resistance marker.
  • pJN3 and pJNY are as expression vectors for introducing bchY and Z genes from BChl b / g-producing bacteria, bciA and bchY / Z gene-deficient mutants endogenous to BChl a-producing bacteria It is necessary to use a drug resistance gene other than the kanamycin resistance gene, streptomycin resistance gene, and spectinomycin resistance gene for selection.
  • kanamycin resistance gene e.g., streptomycin resistance gene, or spectinomycin resistance gene is used to create bciA and bchY / Z gene deletion mutants
  • other drug resistance genes eg, gentamicin resistance gene
  • pJN3 and pJNY can be used.
  • An expression vector containing DNA encoding BchY or BchZ is, for example, excising a target DNA fragment from DNA encoding BchY or BchZ, and the DNA fragment is extracted from the above-mentioned large-scale expression vectors in R. Spheroides and R. Capsulatus. Can be produced by ligating downstream of the promoter.
  • the DNAs encoding BchY and BchZ may be inserted into separate vectors, but it is desirable to insert both into a single vector.
  • the bchY and bchZ genes are present in one operon on the genome of BChl b / g producing bacteria, by subcloning the genome sequence containing both the ORF encoding BchY and the ORF encoding BchZ into an expression vector, It can be designed to express BchYZ from a single promoter polycistronic.
  • the obtained expression vector containing the bchY gene and the bchZ gene can be introduced using an efficient gene transfer system depending on the BChl a-producing bacterium serving as the host, but the host is R. spheroides or R. Capsulatus.
  • gene transfer by conjugation transfer is preferably used, as in the case of producing the bciA and bchY / Z deletion mutants. Specific methods are described in Cryogenic Science, 67: 649-653 (2009).
  • a puc promoter such as pJN3 or pJNY
  • a strain in which the puc promoter is more strongly induced than a wild type strain such as R. Capsulatus DB176, may be used as a recipient.
  • Transformants into which the bchY and bchZ genes derived from BChl b / g-producing bacteria have been successfully introduced can be selected by culturing on an appropriate drug-containing solid medium.
  • the culture is usually performed at a temperature of 20-40 ° C., preferably 25-30 ° C., in a dark (slight) aerobic condition.
  • resistant colonies are obtained in about 1-2 weeks from the start of selection.
  • the obtained mutant strain can be cryopreserved ( ⁇ 70 or 80 ° C. or lower) in a medium containing 5% DMSO or 15% glycerol.
  • the bciA and bchY / Z gene-deficient / BChl b / g-producing bchYZ gene-introduced mutants derived from BChl a-producing bacteria thus obtained are deficient in endogenous BciA and BchYZ having DVR activity.
  • 8V-Chlide a cannot be converted to Chlide a.
  • 8V-Chlide a is converted to BChlide g by the action of BchYZ derived from BChl b / g producing bacteria.
  • BChlide g is converted to BChlide b by the action of endogenous BchF and BchC, and further BChl b is produced by the action of intrinsic BchG (see Fig.
  • the long chain ester group on the C17 position is usually a farnesyl group, but may be a phytyl group, a geranylgeranyl group, a dihydrogeranyl group or a tetrahydrogeranyl group.
  • the present invention also provides a method for producing BChl b, which comprises culturing the BChl b-producing mutant strain of the present invention in a medium and recovering BChl b from the resulting culture.
  • the BChl b production mutant can be cultured by the same method as used in the selection of the bciA and bchY / Z gene-deficient mutants, except that a liquid medium is used instead of the solid medium.
  • a selection agent used for selecting a gene-deficient / transgene-expressing mutant it is desirable to add it to prevent the introduced drug resistance gene from falling off.
  • the concentration of the selective agent may be lower than that used in the selective medium.
  • Extraction of BChlb from the BChlb-producing mutant strain of the present invention can be performed, for example, as follows, but any method known in the art can be used as well.
  • the cultured cells of the BChlb-producing mutant are collected by filtration or centrifugation. Under an anaerobic atmosphere in which nitrogen gas is passed, an acetone / methanol mixed solvent is added to the cells and stirred, and then the liquid phase is recovered, and the same extraction operation is repeated on the residue until no pigment is extracted.
  • the obtained extract is collected, separated and washed with ether and water, and then the ether layer is dehydrated and washed with saturated brine, and further dehydrated by adding anhydrous sodium sulfate.
  • the solvent is distilled off on a rotary evaporator.
  • the residue can be stored frozen by filling with nitrogen gas, or can be stored frozen using diethyl ether or acetone as a solvent. In either case, the container is shielded from light. If light shielding, temperature control, and maintenance of anaerobic conditions are thoroughly implemented, yearly storage is possible.
  • the carotenoid dye is separated and removed from the dye obtained by the above extraction operation by column chromatography, recrystallization or the like, and BChlb is isolated and purified. Sepharose or cellulose can be used as the column, but Sepharose has a higher resolution. Since BChlb has low solubility in nonpolar solvents such as hexane, it can be easily isolated and purified by recrystallization.
  • Example 1 ⁇ Preparation of bciA / bchZ-deficient strain of R. sphaeroides as host>
  • bciA and bchZ gene-deficient mutants hereinafter referred to as dA / Z strains.
  • the method is shown below.
  • R. sphaeroides 2.4.1 strain FEBS Lett 580 (28-29): 6644-6648 2006
  • PYS agar medium J Mol Evol 45 (2): 131-136 1997) containing the antibiotic rifampicin (100 ⁇ g / mL).
  • R. spheroides J001 strain To obtain colonies that spontaneously became rifampicin resistant. This was designated as R. spheroides J001 strain and the parent strain for transformation.
  • R. spheroides J001 strain In order to delete the bciA gene region of R. sphaeroides J001 strain by homologous recombination by a genetic modification method, a plasmid for mutagenesis, pJSCbciAKm, was constructed as follows. Using the genomic DNA of R.
  • This fragment was treated with the restriction enzyme EcoRI and cloned into the SmaI and EcoRI sites of the cloning vector pUC118 (Takara, Japan) to obtain a pUCbciA plasmid.
  • a DNA fragment containing the kanamycin resistance gene neo was obtained by treatment with HindIII and SmaI from pUCKM1 (J Biol Chem 266 (20): 12889-12895 1991), and the cut ends were blunted.
  • the XhoI site inside the bciA gene of pUCbciA was cleaved with a restriction enzyme to make blunt ends.
  • This plasmid fragment and the above neo gene fragment were ligated by a ligation reaction to obtain a pUCbciAKm plasmid.
  • This pUC-bciAKm was treated with restriction enzymes SmaI and XbaI, and the DNA fragment containing the region where the neo gene was replaced with the majority of the bciA gene was converted into the SmaI and XbaI sites of the pJSC vector (Biochemistry 41 (37): 11211-11217 2002).
  • pJSCbciAKm pJSC is a suicide vector, and E. coli JM109 ( ⁇ -pir) strain was used for cloning.
  • pJSC has a mobility site and can be transferred to the J001 strain by transforming it into the Escherichia coli S17-1 ( ⁇ -pir) strain capable of conjugation and then mating with the R. sphaeroides J001 strain. is there. Therefore, S17-1 ( ⁇ -pir) transformed with pJSCbciAKm was mixed with R. sphaeroides J001 strain for conjugation, spotted on PYS agar medium, and incubated at 30 ° C. overnight in the dark.
  • the spots are then scraped off, suspended in PYS liquid medium, spread on PYS agar medium containing rifampicin (100 ⁇ g / mL), kanamycin (25 ⁇ g / mL), and 5% shoecloth, and at 30 ° C in the dark. Incubated. After 5-7 days, a colony of a strain lacking the bciA gene was obtained by homologous recombination.
  • sphaeroides dbciA strain a plasmid for mutagenesis, pJSCbchZSm
  • aadA-F primer sequence: CTGTTCGGTTCGTAAGCTGT (SEQ ID NO: 19)
  • aadA-R primer sequence: CGTCGGCTTGAACGAATTGT (SEQ ID NO: 20)
  • bchYZ-F (sequence: ATGGCATCGCCGCCGACA (SEQ ID NO: 21)
  • bchYZ-R sequence: TAAGACTGACGCCACATGCT (SEQ ID NO: 22)
  • primer set was used for PCR using the genomic DNA of R. sphaeroides J001 strain as a template.
  • a DNA fragment containing a part of the gene and the entire bchZ gene region was obtained.
  • the fragment was cloned into the TA cloning site of T-vector pTA2 (TOYOBO, Japan) to obtain the pTA2bchYZ plasmid.
  • inverse PCR using this plasmid as a template was performed using bciYZ-inf-FI primer (sequence: TCGTTCAAGCCGACG CGTAGAGGAGCATCCGGTT (SEQ ID NO: 23)) and bciYZ-inf-RI primer (sequence: TTACGAACCGAACAG AATGCAGCACCGAGGTCAC (SEQ ID NO: 24)) was used.
  • the underlines of these primer sequences were designed to overlap with the sequences of the aadA-R and aadA-F primers, respectively, for subsequent In-Fusion cloning.
  • the PCR fragment amplified as a result and the fragment containing the aadA gene amplified with the above-mentioned aadA-F and aadA-R were obtained using In-Fusion (registered trademark) HD Cloning Kit (Clontech, USA). Ligation was performed to obtain the pTAbchYZSm plasmid.
  • PCR was performed using the bchYZ-inf-FII primer (sequence: TCGAGCTCGGTACCC TATGAGGGCTCCGAGCTGA (SEQ ID NO: 25)) and bchYZ-inf-RII primer (sequence: CTCTAGAGGATCCCC ACCATGCCCTCCCGATTAAT (SEQ ID NO: 26)). Obtained a DNA fragment containing a region in which most of the bchZ gene was replaced.
  • the underline of the bchYZ-inf-FII and bchYZ-inf-RII primer sequences was designed to overlap with the cut end region of the SmaI site at the multiple clone site of pJSC for subsequent In-Fusion cloning. Therefore, pJSC treated with SmaI and its PCR amplified DNA fragment were ligated by the reaction of In-Fusion kit to prepare pJSCbchYZSm. This pJSCbchYZSm was transformed into E. coli S17-1 ( ⁇ -pir), cultured, mixed with the R. sphaeroides dbciA strain for conjugation, and the bchZ gene was deleted as described above.
  • a PYS agar medium containing rifampicin (100 ⁇ g / mL), streptomycin (50 ⁇ g / mL), and 5% shoe cloth was used. Confirmation of mutagenesis of the obtained colonies is performed by PCR using bchYZ-comf-F primer (sequence: GGCGATCCATCCCTTCTAC (SEQ ID NO: 27)) and bchYZ-comf-R primer (sequence: TATCAGCCATGCTATCCTCC (SEQ ID NO: 28)). In addition, the PCR amplification sequence was determined and further mutation introduction was confirmed. The strain in which the introduction of the mutation was confirmed was named dA / Z.
  • Example 2 ⁇ Preparation method of expression vector pJN7 used for introduction of bchYZ gene of BChl b-producing bacterium B. viridis> Two primers pPucf6 (5'-ata gtcgac ttcactgggattttgcgccc-3 (SEQ ID NO: 29)) and pJr6 (5'-tat ggtacc gatatcaGAGACCccgcGGTCTCggcgccgaccctatgcgactgcattctcgaactgcggtg) ) To obtain a DNA fragment. This DNA fragment contains the promoter Ppuc region and the BsaI restriction enzyme site (uppercase) for use in cloning.
  • pPucf6 5'-ata gtcgac ttcactgggattttgcgccc-3 (SEQ ID NO: 29)
  • pJr6 5'-tat ggtacc gatatca
  • This fragment was treated with BamHI and SalI (underlined), and then an approximately 0.2 kbp fragment was purified (Wizard SV Gel and PCR Clean-Up System: Promega).
  • the broad host range vector pBBR1MCS2 (provided by Professor Carl E. Bauer, Indiana University) was treated with BamHI and SalI. The treated mixture was loaded onto a 1% agarose gel and run at 100 V for 1 hour to purify the approximately 5.3 kbp fragment. Wizard SV Gel and PCR Clean-Up System was used for DNA recovery from the agarose gel.
  • PBBR1MCS2 contains a gene cassette that provides resistance to the antibiotic kanamycin as a marker gene.
  • This pJN6 plasmid has a BsaI restriction enzyme site derived from pBBR1MCS2 in one place in addition to the BsaI restriction enzyme site used for cloning.
  • KOBsaI-f1 (5'-ttcaggcgctcccgaa gatccc gggccgtctcttgg-3 '(SEQ ID NO: 31)
  • KOBsaI-r1 (5'-aagagacggccc gggatc ttcgggagcgc-3' (SEQ ID NO: 32)) was used to amplify the entire region of pJN6 by PCR. This was designated as pJN7pre.
  • Spc2f1 5'-ATA GAGCTCTAGA TAATGCAAGTAGCGTATGC-3 '(SEQ ID NO: 33)
  • Spc2r1 5'-ATA GAGCTCTAGA GCGGATGTTGCGATTACTTCG-3' (SEQ ID NO: 34)
  • the completed pJN7 has been shown to be retained in E. coli and the red non-sulfur bacterium R. capsulatus. Cloning of the gene of interest into this vector utilizes the BsaI restriction enzyme site and can be selected using spectinomycin / streptomycin / kanamycin.
  • Example 3 Preparation of R. sphaeroides mutant producing BChl b>
  • the bciA and bchZ gene-deficient mutant dA / Z obtained in Example 1 and the R. sphaeroides wild strain were grown in PYS medium under dark microaerobic conditions. Using these strains as hosts, bchY and bchZ genes derived from B. viridis were introduced.
  • BvYZ-infu-F1 (sequence: CGAGAAGGGCGGCGCCAGGGCTGCCAGTTACGTTC (SEQ ID NO: 35)
  • BvYZ-infu-R1 (sequence: CTGGGTACCGATATCTCACAGAGCCTGCCCCCCGACA (SEQ ID NO: 36))
  • BvYZ-infu-R1 (sequence: CTGGGTACCGATATCTCACAGAGCCTGCCCCCCGACA (SEQ ID NO: 36)) using the viridis genome as a template, PCR, B.
  • Bilidis-derived bchY and bchZ genes were amplified together (in photosynthetic bacteria, both genes usually overlap, and the start codon of the bchZ gene precedes the stop codon of the bchY gene).
  • the amplified DNA fragment was excised from an agarose gel and purified using NucleoSpin Extract II kit (Macherey-Nagel, Duren, Germany).
  • NucleoSpin Extract II kit Macherey-Nagel, Duren, Germany.
  • This purified DNA fragment containing the B. viridis bchYZ gene was subcloned into the Bsa I restriction site of the pJN7 plasmid prepared in Example 2 using the In-Fusion HD Cloning kit (Clontech, USA), and the plasmid pJ7-BvYZ was Obtained.
  • the Kpn I restriction site is located after the coding region of bchZ.
  • the gentamicin resistance gene aacC1 was obtained from the plasmid pUCGM-star (Biotechniques, 15: 831-834 (1993)) with the following primers, Gm-JN7-F (sequence: CTGCGTGAGATATCGCAACTGGTCCAGAACCTTGA (SEQ ID NO: 37)) and Gm-JN7-R ( The sequence was amplified using GGGAACAAAAGCTGGAAGCTTGCATGCCTGCAGG (SEQ ID NO: 38)).
  • the obtained PCR product containing the aacC1 gene and the Kpn I digested pJ7-BvYZ plasmid were ligated using the In-Fusion HD Cloning kit to obtain pJ7-BvYZ-Gm.
  • This plasmid was introduced into R. sphaeroides bciA and bchZ gene-deficient mutant dA / Z by three-parental conjugation using E. coli Tec5 strain (J Bacteriol. 1983; 154: 580-590 1983) containing a helper plasmid. .
  • Transconjugant was selected on PYS solid medium (J Mol Evol 45 (2): 131-136 1997) containing rifampicin (100 ⁇ g / ml), kanamycin (25 ⁇ g / ml) and gentamicin (10 ⁇ g / ml) .
  • the thus obtained transconjugate expressing B. viridis-derived BchYZ under the control of the puc promoter was named ⁇ bciA / bchZ + BvYZ.
  • Example 4 ⁇ BChlb production by R. sphaeroides ⁇ bciA / bchZ + BvYZ strain>
  • the R. sphaeroides ⁇ bciA / bchZ + BvYZ strain obtained in Example 3 and the strain obtained by introducing the B. viridis-derived bchYZ gene into the wild strain of R. spheroides were each cultured in PYS medium, and the culture solution was centrifuged. Each cell was collected and the pigment was extracted with acetone / methanol (7: 2, vol / vol).
  • BChlb or a derivative thereof having a Qy band in the near-infrared region having a longer wavelength can be provided simply and in large quantities.
  • BChl b or its derivative has the longest absorption wavelength maximum shifted to the near infrared region compared to other chlorin or bacteriochlorin compounds.
  • this as a dye sensitizer, light energy conversion than before is possible.
  • a dye-sensitized solar cell excellent in efficiency can be provided.
  • cancer cells actively divide and proliferate and easily take up fat-soluble porphyrin compounds having a large ⁇ plane, so porphyrin compounds can be used to distinguish cancer cells from normal cells. Attempts have been made to use.
  • BChlb or a derivative thereof has an absorption band in the near-infrared region of a longer wavelength, and thus is useful for deep cancer diagnosis. Furthermore, it has been clarified that chlorophyll molecules also have an anticancer effect, and research is being conducted as photodynamic therapy. Therefore, BChlb or a derivative thereof having a strong absorption band in the near infrared region that is selectively adsorbed to cancer cells can be a useful tool for cancer treatment.
  • This application is based on Japanese Patent Application No. 2014-030085 filed in Japan (filing date: February 19, 2014), the contents of which are incorporated in full herein.

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Abstract

　La présente invention concerne une variante produisant une bactériochlorophylle b obtenue par délétion du gène bciA et du gène bchY et/ou bchZ intrinsèques à une bactérie photosynthétique qui produit une bactériochlorophylle a, et la mise en œuvre d'une manipulation visant à exprimer les gènes bchY et bchZ dérivés d'une bactérie photosynthétique qui produit la bactériochlorophylle b ou la bactériochlorophylle g. La présente invention concerne également un procédé de production de bactériochlorophylle b, qui consiste à mettre en culture ladite variante dans une culture, et à récupérer la bactériochlorophylle b à partir de la culture obtenue.
PCT/JP2015/054552 2014-02-19 2015-02-19 PROCÉDÉ DE PRODUCTION EN MASSE DE BACTÉRIOCHLOROPHYLLE b, ET SOUCHE DE PRODUCTION WO2015125849A1 (fr)

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JP2014003938A (ja) * 2012-06-22 2014-01-16 Ritsumeikan 新規酵素及びそれを用いたバクテリオクロロフィルの製造方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CANNIFFE D.P. ET AL.: "Engineered biosynthesis of bacteriochlorophyll b in Rhodobacter sphaeroides", BIOCHIM.BIOPHYS.ACTA, vol. 37, no. 10, 18 October 2014 (2014-10-18), pages 1611 - 6, XP055221546 *
HARADA J. ET AL.: "Chlorophyllide a oxidoreductase works as one of the divinyl reductases specifically involved in bacteriochlorophyll a biosynthesis", J.BIOL. CHEM., vol. 289, no. 18, 2 May 2014 (2014-05-02), pages 12716 - 26, XP055221547 *
NOMATA J. ET AL.: "A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus", J.BIOL. CHEM., vol. 281, no. 21, 26 May 2006 (2006-05-26), pages 15021 - 8, XP055221545 *
TSUKATANI Y. ET AL.: "An unexpectedly branched biosynthetic pathway for bacteriochlorophyll b capable of absorbing near-infrared light", SCI. REP., vol. 3, 2013, pages 12 17, XP055221541 *
TSUKATANI Y. ET AL.: "Completion of biosynthetic pathways for bacteriochlorophyll g in Heliobacterium modesticaldum: The C8-ethylidene group formation", BIOCHIM.BIOPHYS.ACTA, vol. 27, no. 10, 18 October 2013 (2013-10-18), pages 1200 - 4, XP055221539 *

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