JP2006075097A - Transformant having carotenoid productivity and method for producing carotenoid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new transformant having astaxanthin productivity, to provide a new method for producing the astaxanthin by using the transformant and to provide a new method for producing the carotenoid with which various carotenoids including β-carotene, the astaxanthin, etc., can selectively be produced. <P>SOLUTION: The transformant is obtained by transducing a recombinant vector plasmid comprising a gene encoding phytoene desaturase, a gene encoding lycopene cyclase, a gene encoding β-carotene hydroxylase and a gene derived from a procaryote and encoding β-carotene-C(4)-oxygenase into a photosynthetic bacterium which is a host having carotenoid productivity and transforming the bacterium. The transformant has the carotenoid productivity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel transformant formed by introducing another gene useful for producing a certain carotenoid in a photosynthetic bacterium having carotenoid-producing ability, and a trait introduced with such another gene. The present invention relates to a novel carotenoid production method for producing various carotenoids using a converter.

  Carotenoid is a general term for yellow, red, or purple polyene pigments widely distributed in the animal and plant kingdoms, and is known to have an antioxidant effect on lipids. It is widely used in many fields such as pharmaceuticals and health foods. As for the carotenoid production method, a method mainly by chemical synthesis and a method by extraction from a natural product are known, and typical examples obtained by extraction from a natural product include plants such as carrots. And β-carotene extracted from the astaxanthin extracted from alga bodies of the genus Haematococcus.

In addition, several attempts have been made for methods for efficiently producing such carotenoids using genetic engineering techniques.
For example, E. coli is used as a host, and the gene crtE {enzyme for synthesizing Geranylgeranyl pyrophosphate (GGPP) from Farnesyl pyrophosphate (FPP) (GGPP synthase)}, crtB (Erwinia herbicola) is used as a host. Enzymes that synthesize Phytoene from GGPP (Phytoene synthase)}, crtI (Phytoene desaturase), crtY (Lycopene cyclase), crtZ (β-carotene hydroxylase), and the prokaryote Agrobacterium aurantiacum (Agrobacterium aurantiacum) A method for producing astaxanthin by introducing the gene crtW (β-carotene C (4) oxygenase) or the gene crtO (β-carotene ketolase) derived from the eukaryotic Haematococcus pluvialis has been proposed ( Non-Patent Document 1 and Non-Patent Document 2).

  In addition, there is a method for producing astaxanthin by using a prokaryotic Synechococcus sp. PCC7942 as a host and transforming the Synechococcus by introducing a gene crtO derived from the eukaryotic Haematococcus pluvialis. It has been proposed (see Non-Patent Document 3).

  Furthermore, by cloning a gene group related to the biosynthesis of β-carotene of the prokaryotic photosynthetic bacterium Erythrobacter longus OCh101 and introducing it into the photosynthetic bacterium of the genus Rhodobacter, A method for producing β-carotene using photosynthesis ability has been proposed (see Patent Document 1).

  Also, a method for introducing foreign genes into the green alga Hematococcus by introducing the foreign gene into the eukaryotic green alga Haematococcus by electroporation (electroporation) or transfection using a dendrimer (Transfection). (See Patent Document 2), and as an application thereof, intermediate products such as zeaxanthin and canthaxanthin, which partially deactivate the secondary metabolic pathway of β-carotene to reach astaxanthin, for example, The possibility of biosynthesizing (Cantaxanthin) is described.

  Furthermore, the prokaryotic marine photosynthetic bacterium Rhodovulum sulfidophilum NKPB160471R is used as a host, and the prokaryotic photosynthetic bacterium Erythrobacter longus OCh101 is derived. In addition to introducing the genes crtI and crtY related to β-carotene biosynthesis, the genes crtO and crtZ derived from the eukaryotic Haematococcus pluvialis NIES144 were introduced to establish an astaxanthin production system in photosynthetic bacteria. (See Non-Patent Document 4). However, this paper reports that biosynthesis did not proceed beyond β-carotene.

Japanese Patent Application Laid-Open No. 08-89,241 Japanese Patent Laid-Open No. 2004-33,070 N. Misawa & H. Shimada, Journal of Biotechnology, Vol.59, 169-181 (1998) Kajiwara S, K.T., Saito T, Kondo K, Ohtani T, Nishio N, Nagai S & Misawa N, Plant Mol. Biol., Vol. 29, 343-352 (1995) M. Harker & J. Hirschberg, FEBS Letters 404, 129-134 (1997) Fujiwara, January 2003 Master's Thesis, Tokyo University of Agriculture and Technology

  However, any of the methods proposed and reported so far are not necessarily suitable as methods for selectively mass-producing a wide variety of carotenoids. For example, non-patent documents In the method described in No. 1 and Non-Patent Document 2, many genes need to be introduced, and since Escherichia coli does not have a carotenoid synthesis system, the content of precursors for carotenoid synthesis is low, and carotenoids such as astaxanthin. Is unsuitable as a method for mass-producing, and in the method described in Non-Patent Document 3, the amount of astaxanthin synthesized is 0.015% by weight relative to the dry alga body weight, and 0.143 mg / L per culture solution. Furthermore, the method described in Patent Document 1 has not yet been synthesized after β-carotene. Furthermore, the methods described in Patent Document 2 and Non-Patent Document 4 are not used. Iterator carotenoids, not specifically established as a method for producing astaxanthin.

  Accordingly, the present inventors have developed a new transformant having the ability to produce carotenoids for biosynthesis of various carotenoids including β-carotene and astaxanthin, and production of new carotenoids using the transformant. As a result of diligent examination of the method, a photosynthetic bacterium capable of producing carotenoid was used as a host, and a gene encoding phytoene desaturase (crtI) and a gene encoding lycopene cyclase (Lycopene cyclase) were used in the host photosynthetic bacterium. (CrtY), derived from a prokaryotic organism encoding a gene (crtZ) encoding β-carotene hydroxylase and β-carotene C (4) -oxygenase (β-carotene C (4) oxygenase) Recombinant vector plasmid containing one or more specific genes selected from genes (crtW) By using the obtained transformant, the precursor for carotenoid synthesis inherent in photosynthetic bacteria is used, and various carotenoids up to astaxanthin can be selectively used depending on the type of the introduced gene. The present invention has been completed by finding that it can be biosynthesized.

  Accordingly, an object of the present invention is to provide a novel transformant having astaxanthin-producing ability.

  Another object of the present invention is to provide a novel astaxanthin production method for producing astaxanthin using the novel transformant capable of producing astaxanthin.

  Another object of the present invention is to provide a novel carotenoid production method capable of selectively producing various carotenoids such as β-carotene and astaxanthin.

  That is, the present invention relates to a gene encoding phytoene desaturase, a gene encoding lycopene cyclase, a gene encoding β-carotene hydroxylase, β-carotene hydroxylase, A recombinant vector plasmid containing a prokaryotic gene encoding carotene-C (4) -oxygenase (β-carotene C (4) oxygenase) is introduced into a host photosynthetic bacterium capable of producing carotenoids and transformed. It is a transformant having carotenoid-producing ability obtained by

  In addition, the present invention uses a mutant of Rhodovulum sulfidophilum, a marine photosynthetic bacterium in which the carotenoid synthesis pathway after Neurosporene is inactivated, as a host, and encodes phytoene desaturase (Phytoene desaturase). A recombinant vector plasmid containing the gene crtI, a recombinant vector plasmid containing the gene crtI and the gene crtY encoding lycopene cyclase, the gene crtI and the gene crtY and β-carotene hydroxylase (β- selected from a recombinant vector plasmid comprising a gene crtZ encoding carotene hydroxylase and / or a gene crtW encoding β-carotene C (4) -oxygenase derived from prokaryotes. Any recombinant vector plasmid introduced into the host Te is transformed carotenoids method of producing, characterized in that to produce the desired carotenoid by using the obtained transformant.

  In the present invention, the photosynthetic bacterium having the ability to produce carotenoid as a host is preferably a prokaryotic organism that grows faster than Haematococcus pluvialis having the ability to produce astaxanthin. Various photosynthetic bacteria such as certain Rhodobulum sulfide phyllum, Erythrobacter genus, Lodibacter genus, Rhodobulum genus can be exemplified, but in constructing a synthetic pathway to astaxanthin, marine photosynthetic bacteria are more preferable. It is desirable that the mutant be derived from rhodobulum sulfide phyllum and inactivate the carotenoid synthesis pathway after Neurosporene. By using rhodobulum sulfide phyllum, in which the carotenoid synthesis pathway after Neurosporene was inactivated, the synthesis pathway from neurosporene to spheroidene in rhodobulum sulfide phyllum was blocked. It becomes easy to construct a synthetic route from neurosporene to astaxanthin.

  A method for obtaining a mutant of rhodobulum sulfide filam, which is such a marine photosynthetic bacterium, is not particularly limited. For example, a predetermined concentration of EMS (in a cell suspension adjusted to a predetermined cell concentration) Ethyl methanesulfonate) and then incubated at room temperature for several minutes, then the resulting cells were washed with a phosphate buffer, further washed with a medium for photosynthetic bacteria, then inoculated on an agar plate and cultured, By selecting only green strains from among the grown strains and collecting them, a rhodobulum sulfide filam mutant can be obtained. The rhodobulum sulfidophilum mutant used in Example 1 of the present invention is named Rhodovulum sulfidophilum NKPB160471RM, and received the receipt number “ Deposited as “NITE AP-21”.

  In addition, a recombinant vector plasmid introduced into such a photosynthetic bacterium as a host includes a gene encoding phytoene desaturase, a gene encoding lycopene cyclase, a gene encoding β-carotene hydroxylase, and β-carotene. It is preferably a recombinant vector plasmid containing any one or more genes selected from prokaryotic genes encoding -C (4) -oxygenase, and more specifically, phytoene desaturase A recombinant vector plasmid containing the encoding gene crtI, a recombinant vector plasmid containing the gene crtI and the gene crtY encoding lycopene cyclase, a gene encoding β-carotene hydroxylase in addition to the gene crtI and the gene crtY crtZ and / or prokaryotic Objects from the β- carotene -C (4) - may be from a recombinant vector plasmid containing the genes crtW encoding oxygenase.

  Regarding the microorganisms for obtaining the above genes crtI, crtY and crtZ, since the photosynthetic bacterium used in the present invention is preferably a prokaryotic marine photosynthetic bacterium, preferably a gene derived from a prokaryote, like the gene crtW More specifically, more specifically, Erwinia herbicola, Agrobacter Aurant Akamu {or Paracoccus sp. MBIC 1143}, cyanobacteria, and others such as erythrobacter genus, rhodobacter genus, rhodobulum genus, etc. A photosynthetic bacterium can be exemplified. By using a prokaryotic organism together with a microorganism that obtains these genes crtI, crtY, crtZ, and crtW and a photosynthetic bacterium that has the ability to produce carotenoids of a host into which these genes crtI, crtY, crtZ, and crtW are introduced. Since the frequency of codon usage in synthesizing enzyme (protein) synthesis is similar, it is considered that as a result, the enzyme is synthesized efficiently and carotenoid synthesis proceeds.

  In the present invention, the genes crtI, crtY, crtZ and crtW for preparing the recombinant vector plasmid are known from various microorganisms such as the alkaline SDS method and the triton lysozyme method. It can be obtained by extracting by a method and then amplifying by a PCR method using a specific primer. Further, as a method for constructing a recombinant vector plasmid using these obtained genes crtI, crtY, crtZ and crtW, a method based on a ligation reaction using a DNA ligase known so far can be employed. Furthermore, methods for introducing the constructed recombinant vector plasmid into a photosynthetic bacterium having the ability to produce carotenoids as a host for transformation are also known, for example, electroporation, conjugation transfer, chloride, Various methods such as the calcium method can be employed. Furthermore, the culture of the obtained transformant has been known so far, such as the same method as the method for culturing photosynthetic bacteria before transformation. Can be adopted.

  In the present invention, a particularly preferred transformant is a mutant of rhodobulum sulfide filam, which is a marine photosynthetic bacterium in which the carotenoid synthesis pathway after Neurosporene is inactivated (reception number: NITE AP-21). ), A prokaryotic gene crtI encoding phytoene desaturase, a prokaryotic gene crtY encoding lycopene cyclase, a prokaryotic gene crtZ encoding β-carotene hydroxylase, and β-carotene-C ( 4) A prokaryotic gene crtW encoding oxygenase and a plasmid obtained by introducing a recombinant vector plasmid containing puf as a promoter. This transformant has the ability to produce carotenoids up to astaxanthin, and is named Rhodovulum sulfidophilum NKPB160471RM (pRKY415WZ). It was accepted by the National Institute of Technology and Evaluation Center for Biotechnology as of September 7, 2004. Deposited under the number “NITE AP-22”.

  In the carotenoid production method of the present invention, a lycopene (Lycopene) as a final product and a recombinant vector plasmid containing genes crtI and crtY were introduced from a transformant introduced with a recombinant vector plasmid containing the gene crtI. From the transformant, β-carotene (β-carotene) as a final product, and from a transformant into which a recombinant vector plasmid containing genes crtI, crtY and crtZ has been introduced, zeaxanthin as a final product. From the transformant in which a recombinant vector plasmid containing the genes crtI, crtY and crtW has been introduced, canthaxanthin (Canthaxanthin) as the final product, and further the recombination containing the genes crtI, crtY, crtZ and crtW Astaxanthin as a final product from transformants introduced with vector plasmids It can be produced.

  In the present invention, a photosynthetic bacterium having the ability to produce carotenoids is used as a host, and the photosynthetic bacterium encodes a gene encoding phytoene desaturase (crtI), a gene encoding lycopene cyclase (crtY), and β-carotene hydroxylase. Introducing a recombinant vector plasmid containing one or more specific genes selected from a gene (crtZ) and a prokaryotic-derived gene (crtW) encoding β-carotene-C (4) -oxygenase Various carotenoids up to astaxanthin can be selectively synthesized according to the type of gene introduced by transforming and using the resulting transformant.

  The photosynthetic bacterium having the ability to produce carotenoids used in the present invention itself has a metabolic pathway for synthesizing a precursor of carotenoid synthesis, and further has a growth rate compared to Haematococcus prubiaris and other microalgae. Various carotenoids can be produced industrially advantageously at high speed.

  Hereinafter, preferred embodiments of the present invention will be described in detail based on examples and comparative examples.

[Example 1]
[Step 1] Preparation of Paracoccus sp. MBIC1143 total DNA The total DNA was grown in a prokaryotic Paracoccus sp. MBIC 1143 in RCVB liquid medium (1 liter, pH 6.8) having the composition shown in Table 1. It was prepared from the obtained microbial cells. After collection by centrifugation, the suspension was suspended in 10 times the volume of the protease K buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.4, 50 mM EDTA), and protease K was added to a concentration of 0.1 mg / ml. 10 wt% -sodium lauryl sulfate (SDS) was added to a final concentration of 0.5 wt%, mixed well, and then digested at 37 ° C. for 1 hour. After extraction with a phenol: chloroform (1: 1) solution three times and a chloroform extraction once, 1/10 volume of 3M sodium acetate (pH 7.4) and 2.5 times the volume of the recovered aqueous solution The total DNA was collected by centrifugation. After rinsing with 70% -ethanol, the supernatant was removed, air-dried, and dissolved in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA).

[Step 2] Obtaining crtW (Accession No. D58420) and crtZ (Accession No. D58420) by PCR method Using the genomic DNA (10 ng / ul) obtained in the above step 1 as a template, AmpliTaq Gold (Applied Biosystems) PCR was performed using the same product. In this PCR reaction, the conditions of 1 cycle were 95 ° C. for 30 seconds for heat denaturation, 30 seconds for annealing, 57.2 ° C. for 1 minute for annealing, 59.4 ° C. for 1 minute for annealing, and 1 minute for crtZ, and Elongation was performed at 72 ° C. for 1 minute for 30 cycles. Primers specific to crtW and crtZ shown in Table 2 were used.

[Step 3] Construction of Recombinant Vector Plasmid Next, restriction enzymes KpnI (TaKaRa) and Sse8387I (TaKaRa) were used at a rate of 1 U with respect to 1 ug of DNA, respectively, at 37 ° C. for 1 hour. Each of crtW and crtZ is excised by digestion and then ligation kit (ligation kit ver.2 manufactured by TaKaRa) is used, and then crtW and crtZ excised with pRKY415 digested with KpnI and Sse8387I (puf promoter, crtI derived from Erythrobacter longus OCh101) Ligation reaction with a vector in which (Accession No. D83514) and crtY (Accession No. D83513) are incorporated; Takeyama, H. et al., J. Mar. Biotechnol. 4, pp 224-229 (1996)) Vector: Insert = 1: 10) was performed to construct a recombinant vector plasmid (pRKY415WZ) incorporating crtI, crtY, crtW, and crtZ derived from the target prokaryote.
Here, the vector (pRKY415) used for the construction of the recombinant vector plasmid and the constructed recombinant vector plasmid (pRKY415WZ) are shown in FIG.

[Step 4] Rhodovulum sulfidophilum
Preparation of a mutant of 1. Ethyl sulfonic acid (EMS) is added to 1.5 ml of rhodobulum sulfide filam NKPB160471R adjusted to a cell concentration of 1 × 10 ⁹ cells / ml to a concentration of 4% by weight at room temperature. Incubate for 5 minutes, then wash the cells 4 times with phosphate buffer (pH 7.0) and 2 times with photosynthetic bacteria medium (RCVB medium), then put on agar plates (containing Rifampicin 80 μg / ml) Inoculated and cultured at 32 ° C. in the dark, and a green strain (mutant) was collected from the resulting culture.
This mutant of rhodobulum sulfide filam is named Rhodovulum sulfidophilum NKPB160471RM, and is dated September 7, 2004 by the inventor Satoshi Matsunaga, the Center for Patent Biological Deposits, National Institute of Technology and Evaluation. Was deposited under the deposit number “NITE AP-21”.

[Step 5] Transformation of Rhodobulum Sulfide Filam The recombinant vector plasmid (pRKY415WZ) obtained in Step 3 above was transformed into E. coli S17 by electroporation (2.5 V, 25 uFD, 200Ω, Gene Pulser, Biorad). -1 introduced. The obtained E. coli S17-1 transformant (1.0 × 10 ¹⁰ cells / ml) and rhodobulum sulfide phyllum NKPB160471RM (1.0 × 10 ¹⁰ cells / ml) were mixed at a ratio of 1:10, and the RCVB plate was mixed. And incubated at 32 ° C. under dark conditions for 24 hours.
The resulting transformant of rhodobulum sulfide phyllum was named Rhodovulum sulfidophilum NKPB160471RM (pRKY415WZ). On September 7, 2004 by the inventor, Makoto Matsunaga. Deposited to the Patent Biological Deposit Center under the deposit number “NITE AP-22”.

[Step 6] Extraction and measurement of carotenoid The transformant {NKPB160471RM (pRKY415WZ)} of rhodobulum sulfide phyllum mutant (NKPB160471RM) obtained in Step 5 above was added to RCVB liquid medium at 30 μg / ml kanamycin. After inoculating 10 L of RCVBN liquid medium prepared by addition and culturing for 8 days at 32 ° C. under aerobic conditions, the cells were collected by centrifugation and freeze-dried.

  Extract the dye by adding 5 ml of extraction solvent acetone to 0.5 g of the resulting freeze-dried cells, and use the resulting acetone extract as a Waters Symmetry C18 column (150 mm × 3.9 mm; 5 μm). And HPLC analysis (1 ml / min, 40 ° C.) using acetonitrile: methanol: 2-propanol (90: 6: 4) as an elution solvent.

A peak with a maximum absorption of 480 nm was obtained at a retention time of 3.7 minutes. When fractionated and subjected to mass spectrometry, a molecular weight of 597 was shown, which was in good agreement with a standard product of astaxanthin (manufactured by Sigma) and produced astaxanthin I confirmed that
The carotenoid composition in the acetone extract obtained in Example 1 is shown in Table 5 below.

[Example 2]
Rhodovulum sulfidophilum (Rhodovulum sulfidophilum) in the same manner as in Example 1 except that the above-described pRKY415 (a vector in which crfI and crtY derived from Erythrobacter longus OCh101 are incorporated) was used as a recombinant vector plasmid. NKPB160471RM was transformed to obtain a transformant {NKPB160471RM (pRKY415)}. The obtained transformant {NKPB160471RM (pRKY415)} was cultured in the same manner as in Example 1, and then carotenoids were extracted and measured. The results are shown in Table 5.

[Example 3]
In Example 1 above, a recombinant vector plasmid (pRKY415Z) incorporating crtI, crtY, and crtZ was constructed using the gene crtZ excised in step 3 above, in the same manner as in Example 1 above. A transformant {NKPB160471RM (pRKY415Z)} was prepared and cultured in the same manner as in Example 1, and then carotenoids were extracted and measured. The results are shown in Table 5.

[Example 4]
In Example 1 above, a recombinant vector plasmid (pRKY415W) incorporating crtI, crtY, and crtW was constructed using the gene crtW excised in step 3 above, in the same manner as in Example 1 above. A transformant {NKPB160471RM (pRKY415W)} was prepared and cultured in the same manner as in Example 1, and then carotenoids were extracted and measured. The results are shown in Table 5.

[Comparative Example 1]
Rhodobulum sulfide phyllum NKPB160471R itself, which has not been subjected to mutation and transformation procedures, was cultured in the same manner as in Example 1 above, and carotenoids were extracted and measured. The results are shown in Table 5.

[Comparative Example 2]
The rhodobulum sulfide phyllum mutant NKPB160471RM (accession number: NITE AP-21) obtained in Step 4 of Example 1 was cultured in the same manner as in Example 1 and carotenoids were extracted and measured. It was. The results are shown in Table 5.

[Comparative Example 3]
[Step 1] Extraction of crtO and crtZ genes from Haematococcus pluvialis Using a basal medium (1 liter, pH 6.8) with the composition shown in Table 3, H. pluvialis is subjected to a 12-12 hour light-dark cycle (20 μmol photons / m ² / s) Incubated for 4 days. Add sodium acetate and iron sulfate to a final concentration of 45 mM and 450 μM, respectively, and stir with a stirrer for 8 hours to promote cyst formation of algae with intense light (125 μmol photons / m ² / s) went. Total-RNA was extracted from the cystized H. pluvialis using the hot phenol method, and mRNA was purified using an Oligo dT column (TaKaRa).

[Step 2] Amplification of crtO and crtZ Using the obtained mRNA as a template, reverse transcription was performed using RT-PCR kit ReverTra Dash (TOYOBO). At this time, Random primer was used as a primer. As a pretreatment for the reverse transcription reaction, a mixed solution of mRNA and primer was incubated at 70 ° C. for 10 minutes.
Using cDNA obtained by reverse transcription reaction at 30 ° C for 10 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes, and 4 ° C for 5 minutes, after RNase treatment at 37 ° C for 20 minutes, spin column After purification, PCR was performed using AmpliTaq Gold (Applied Biosystems) as the DNA polymerase. The PCR reaction was performed by heat denaturation; 95 ° C. for 30 seconds, annealing; 55 ° C. for 1 minute, extension; 72 ° C. for 1 minute for 30 cycles. Primers specific to crtO and crtZ shown in Table 4 [crtO and crtZ-specific RT-nested PCR primers (with RBS and restriction enzyme sites)] were used.

[Step 3] Construction of recombinant vector plasmid
The crtO and crtZ obtained by RT-PCR from H. pluvialis NISE 144 were TA cloned using pGEM-T Easy Vector System I from Promega. CrtO or crtZ was excised from pGEM by restriction enzyme digestion (37 ° C, 1 hour) using EcoR I (1U for 1ug of DNA), and ligation of crtO and crtZ (equimolar amount, Takara ligation kit ver.2) (16 ° C., 30 minutes). Next, ligation of the ligation product (crtOZ) and the recombinant vector plasmid pRKY415 digested with KpnI (1 U against 1 ug of DNA) (vector: insert = 1: 10, Takara ligation kit ver.2), A recombinant vector plasmid pRKY415OZ incorporating crtI, crtY, crtO and crtZ was constructed.

[Step 4] Transformation of rhodobulum sulfide phyllum The recombinant vector plasmid pRKY415OZ obtained in the above step 3 was transformed into E. coli S17-1 by electroporation (2.5 V, 25 uFD, 200Ω, Gene Pulser manufactured by Biorad). Introduced. The obtained transformant (1.0 × 10 ¹⁰ cells / ml) was mixed with rhodobulum sulfide filam NKPB 160471R (1.0 × 10 ¹⁰ cells / ml) at a ratio of 1:10, spotted on an RCVB plate, and darkened for 24 hours. Incubate at 32 ° C under conditions.

[Step 5] Carotenoid Extraction and Measurement RCVBN prepared by adding the rhodobulum sulfide filam transformant {NKPB 160471R (pRKY415OZ)} obtained in Step 4 above to RCVB liquid medium with 30 μg / ml kanamycin. After culturing in the medium, the cells were collected by centrifugation and freeze-dried. After the dye was extracted with acetone, HPLC analysis (1 ml / min, 40 ° C.) was performed with acetonitrile: methanol: 2-propanol (90: 6: 4) using a Waters Symmetry C18 column (150 mm × 3.9 mm; 5 μm). Only the β-carotene peak was shown, and the activity of the introduced CrtO and Z was not confirmed. The results are shown in Table 5.

  The novel transformant having the ability to produce carotenoid and the method for producing the carotenoid of the present invention effectively use the precursor for carotenoid synthesis possessed by the photosynthetic bacterium of the host, and the newly introduced gene type It is possible to selectively biosynthesize various carotenoids up to astaxanthin according to the above, and it shows the possibility of selective synthesis of various carotenoids, contributing to the future industrial production of carotenoids. is there.

FIG. 1 is a conceptual diagram showing the vector (pRKY415) used for the construction of the recombinant vector plasmid and the constructed recombinant vector plasmid (pRKY415WZ) in Example 1.

Claims (5)

  A gene encoding phytoene desaturase, a gene encoding lycopene cyclase, a gene encoding β-carotene hydroxylase, and β-carotene-C (4) -Carotenoids obtained by transforming by introducing a recombinant vector plasmid containing a prokaryotic gene encoding oxygenase (β-carotene C (4) oxygenase) into a host photosynthetic bacterium capable of producing carotenoids A transformant having a production ability.   The transformant according to claim 1, wherein the host photosynthetic bacterium is a marine photosynthetic bacterium Rhodovulum sulfidophilum.   The transformant according to claim 2, wherein the host marine photosynthetic bacterium is a mutant of rhodobulum sulfide phyllum in which the carotenoid synthesis pathway after Neurosporene has been inactivated.   The gene encoding phytoene desaturase is crtI derived from prokaryotes, the gene encoding lycopene cyclase is crtY derived from prokaryotes, and the gene encoding β-carotene hydroxylase is crtZ derived from prokaryotes. The transformant according to any one of claims 1 to 3, wherein the gene encoding β-carotene-C (4) -oxygenase is prokaryotic-derived crtW. A mutant of Rhodovulum sulfidophilum, a marine photosynthetic bacterium with inactivated carotenoid synthesis pathway after Neurosporene, is used as a host, and the mutant of Rhodobulum sulfidophilum of this host is used as a phytoene. A recombinant vector plasmid containing a gene crtI encoding a desaturase (Phytoene desaturase), a recombinant vector plasmid containing the gene crtI and a gene crtY encoding a lycopene cyclase, the gene crtI and the genes crtY and β A gene crtZ encoding β-carotene hydroxylase and / or a gene crtW encoding β-carotene C (4) -oxygenase derived from prokaryotes Any one selected from recombinant vector plasmids A method for producing carotenoids, characterized by producing a carotenoid according to the type of gene introduced using a transformant obtained by introducing a recombinant vector plasmid.

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