# Patent

## Title
Method for production of carotenoid-synthesizing microorganism and method for production of carotenoid

## Abstract

A method for producing a carotenoid comprising the steps of cultivating a cell transformed with a DNA sequence comprising a DNA sequence depicted in anyone of SEQ ID NOs: 2-7 or a cell transformed with a vector having a DNA sequence depicted in anyone of SEQ ID NOs: 2-7 under proper culture conditions and isolating the carotenoid from the cell or the culture.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a 35 U.S.C. §371 National Stage patent application of International patent application PCT/JP06/318904, filed on Sep. 25, 2006, which claims priority to Japanese patent application JP 2005-315070, filed on Oct. 28, 2005. 
TECHNICAL FIELD
The present invention relates to a method of preparing a carotenoid synthesizing microorganism and a method of producing carotenoids.
BACKGROUND ART
The present invention relates to DNA chains which are useful for synthesis of carotenoids suitably used for reviving the colors of farm-raised fish such as sea bream, shrimp and salmon, and hen eggs, and for synthesis of carotenoids such as astaxanthin which is applicable as a coloring agent or antioxidant to foods, and to methods of producing carotenoids such as astaxanthin utilizing microorganisms incorporating such DNA chains.
In the natural word, over 600 of different carotenoids have been identified from plants, microorganisms and the like. Industrially useful carotenoids are generally produced by chemical synthesis processes for which possibility of undesired actions such as contamination of synthesis auxiliary materials is feared. In addition, tastes of consumers tend to lean toward naturally-occurring carotenoids. However, there is a limit to extraction from plants and the like natural products, and an effective industrial process is not entirely established. As a production method of naturally-occurring carotenoids, microbial fermentation methods have been reported in some cases, however, none of such cases enable production of carotenoids in an amount which is enough for economical industrial production. Likewise the cases of carotenoids, when trying to produce a functional substance from a microorganism, one will choose a microorganism which serves as a host of fermentation by broad screening. Then, in many cases, through classical mutation and breeding using a chemical treatment agent, a highly productive strain is isolated and subjected to production or research, because a production amount from a wild-type of carotenoid producing microorganism is usually small.
As a microorganism that produces useful carotenoid, Yokoyama et al. reported Agrobacterium (later, reclassified into bacteria belonging to Paracoccus) marine bacteria (Non-patent document 1). These strains are characterized by synthesizing astaxanthin which is a functional carotenoid in high content. As described above, a production amount of astaxanthin or the like of Paracoccus bacterial can be increased through mutation process, and a strain TSN18E7 with improved production amount (see Japanese Patent Laid-Open Publication 2005-58216) is deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the number of FERM P-19746.
A carotenoid biosynthesis pathway is made up of various enzymes, and genes encoding such enzymes have been analyzed by many researches. In a typical pathway, for example, carotenoid is synthesized in its early stage by an isoprenoid biosynthesis pathway which is shared by steroid and terpenoid, starting from mevalonic acid which is a basic metabolite. Farnesyl pyrophosphate having 15 carbons (C15) generating through the isoprenoid basic synthesis system is condensed with isopentenyl diphosphate (IPP) (C5), to give geranylgeranyl diphosphate (GGPP) (C20). Then through condensation of two molecules of GGPP, colorless phytoene which is the first carotenoid is synthesized. The phytoene is then converted into lycopene through a series of unsaturation reactions, and then the lycopene is converted into β-carotene through a cyclization reaction. Then, a hydroxyl group and a keto group are introduced into the β-carotene, which leads synthesis of various xanthophylls represented by astaxanthin (FIG. 1).
From these gene level findings, studies intended to improvement of carotenoid synthesis with the use of genetic recombination technique have been made. See Chia-wei Wang et al., Biotechnol. Prog., 16: 922-926 (2000); Claudia Schmidt-Dannert et al., Nat. Biotechnol., 18: 750-753 (2000); Daisuke Umeno et al., Appl. Environ. Microbiol., 69: 3573-3579 (2003), for example. In these studies, Escherichia coli that does not synthesize carotenoid is used as a host, so that it would be difficult to apply these studies to industrial production because of their low productivity of carotenoid. In other report, increase in carotenoid synthesis amount is realized by introducing a carotenoid gene into a bacterium that produces carotenoid (Patent document 1). However, it would be still difficult to apply such prepared gene recombinant strain to industrial production because of its low amount of carotenoid synthesis.

    [Non-patent document 1] Yokoyama, A. H. Izumida, and W. Miki, Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum, Biosci. Biotechnol. Biochem., 58: 1842-1844 (1994).
    [Non-patent document 2] Norihiko Misawa, Yoshiko Satomi, Keiji Kondo, Akihiro Yokoyama, Susumu Kajiwara, Tochiko Saito, Takeshi Ohtani, and Wataru Miki, Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level, J., Bacteriology 177: 6575-6584 (1995).
    [Non-patent document 3] Eric A. Johnson, and William A. Schroeder, Microbial Carotenoids, Advances in Biochemical Engineering Biotechnology, 53: 119-178 (1995).
    [Non-patent document 4] P. C. Lee, and Schmidt-Dannert, Metabolic engineering towards biotechnological production of carotenoids in microorganism, 60:1-11 (2002).
    [Non-patent document 5] Kovach, M. E. et al., GENE166, 175-176 (1995).
    [Non-patent document 6] R. Simon, U. Priefer, and A. Puhler, A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria, BIO/TECHNOLOGY, 1: 784-791 (1983).
    [Non-patent document 7] Cedric Y. Szpiper, Michel Faelen, and Martine Couturier, Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1, J. Bacteriology, 183: 2101-2110 (2001).
    [Patent document 1] Japanese translation of PCT application JP-A 2004-527265
    [Patent document 2] Japanese Patent Publication No. 3403381
    [Patent document 3] Japanese Patent Application No. 2005-106045


SUMMARY OF THE INVENTION
Means to be Solved by the Invention
It is an object of the present invention to prepare a microorganism which allows carotenoid production in industrial production scale. It is also an object of the present invention to produce carotenoids using a novel carotenoid producing strain and to provide the carotenoids.
Means for Solving the Problem
Through diligent efforts for solving the aforementioned problems, the present inventors found that a synthesis amount of carotenoid is increased by cloning a carotenoid synthesis gene of Paracoccus sp. strain MBIC1143 which is a carotenoid synthesis bacterium; recombining the carotenoid synthesis gene into an appropriate plasmid vector; introducing the plasmid vector harboring the carotenoid synthesis gene inserted therein into a wild-type strain, a mutant strain with increased carotenoid synthesis ability as a result of mutation treatment, or a carotenoid synthesis tolerant strain of Paracoccus sp. by a gene transduction technique such as conjugational transfer; and letting a carotenoid synthase encoded by the carotenoid synthesis gene express.
In other words, the present inventors found that a carotenoid content such as astaxanthin is dramatically increased by introducing into Paracoccus sp. strain MBIC1143, a DNA chain selected from the group consisting of (a) DNA chain encoding a polypeptide having such an enzymatic activity that converts a methylene group at 4 position in β-ionone ring into a keto group (crtW), (b) DNA chain encoding a polypeptide having such an enzymatic activity that adds one hydroxyl group to a carbon at 3-position of 4-keto-β-ionone ring and/or at 3-position of β-ionone ring (crtZ), (c) DNA chain encoding a polypeptide having such an enzymatic activity that converts lycopene into β-carotene (crtY) (d) DNA chain encoding a polypeptide having such an enzymatic activity that converts phytoene into lycopene (crtI), (e) DNA chain encoding a polypeptide having prephytoene synthase activity (crtB), and (f) DNA chain encoding a polypeptide having geranylgeranyl diphosphate synthase activity (crtE), and completed the present invention.
(1) A method of increasing generation of carotenoid, wherein a DNA chain selected from the group consisting of the (a), (b), (c), (d), (e) and (f) is introduced into a carotenoid producing microorganism such as Paracoccus sp., and the resultant transformed microorganism is cultured in a culture medium.
(2) The method of increasing generation of carotenoid according to (1), wherein the polypeptide having a carotenoid synthesis activity is a polypeptide of (i) or (ii) below.
(i) Polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6 or 7.
(ii) Polypeptide having an amino acid sequence which is substantially homologous to an amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6 or 7.
The present invention also relates to carotenoid producing methods of (3) to (4) below.
(3) A method of producing a carotenoid, wherein a DNA chain selected from the group consisting of the (a), (b), (c), (d), (e) and (f) is introduced into a carotenoid producing microorganism such as Paracoccus sp., and the resultant transformed microorganism is cultured in a culture medium.
(4) The method of producing carotenoid according to (3), wherein the polypeptide having a carotenoid synthesis activity is a polypeptide of (i) or (ii) below.
(i) Polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6 or 7.
(ii) Polypeptide having an amino acid sequence which is substantially homologous to an amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6 or 7.
The present invention also relates to a transformation method, wherein a DNA chain selected from the group consisting of the above (a), (b), (c), (d), (e) and (f) or a plasmid vector having the DNA chain inserted therein is introduced into a carotenoid producing microorganism such as Paracoccus sp. The present invention also relates to a microorganism having improved carotenoid productivity by introducing a plasmid into a cell, of which cell growth is not influenced by the replication of the plasmid occurring in a cell by the introducing a plasmid vector, and/or by carotenoids produced by carotenoid synthesis gene encoded by the plasmid
Effect of the Invention
According to the present invention, there are provided a DNA chain which significantly improves a production amount in biosynthesis of carotenoid by microorganism, and a method which involves introduction of the DNA chain into a carotenoid producing microorganism and expression thereof, thereby increasing an amount of carotenoid produced by the microorganism several times.


BEST MODE FOR CARRYING OUT THE INVENTION
In the following, the present invention will be described in detail.
As is described in detail in the section of “conventional art”, microorganisms such as Escherichia coli are enabled to produce useful carotenoids such as astaxanthin, zeaxanthin, β-carotene and lycopene as a result of introduction of carotenoid biosynthesis gene from carotenoid producing bacteria such as marine bacteria Paracoccus sp. and the like. On the other hand, in order to compete with costs of organic synthesis methods, it is necessary to increase a production amount of carotenoid as much as possible. The DNA chain of the present invention is selected from the group consisting of (a) DNA chain encoding a polypeptide having such an enzymatic activity that converts a methylene group at 4 position in β-ionone ring into a keto group (crtW), (b) DNA chain encoding a polypeptide having such an enzymatic activity that adds one hydroxyl group to a carbon at 3-position of 4-keto-β-ionone ring and/or at 3-position of β-ionone ring (crtZ), (c) DNA chain encoding a polypeptide having such an enzymatic activity that converts lycopene into β-carotene (crtY), (d) DNA chain encoding a polypeptide having such an enzymatic activity that converts phytoene into lycopene (crtI), (e) DNA chain encoding a polypeptide having prephytoene synthase activity (crtB), and (f) DNA chain encoding a polypeptide having geranylgeranyl diphosphate synthase activity (crtE), and is extremely useful for increasing a production amount of carotenoid, in particular, astaxanthin. By raising an expression level of gene of enzyme or the like through current advanced genetic engineering techniques, a production amount of a protein encoded by the gene can be increased relatively easily. In metabolic concept, there is a report that introduction of IPP isomerase gene located on upstream side of the carotenoid synthesis system significantly increases production of carotenoid in an expression cell (Patent document 2). The success in increasing a carotenoid production amount by introduction of IPP isomerase gene is attributed to the fact that the upstream metabolic pathway up to FPP (FIG. 1) is broadened by the introduction, resulting in increase in supply amount of FPP and hence increase in amount of carotenoid.
However, keeping the production of carotenoid, in particular, of astaxanthin which is one of the objects of the present invention in mind, subsequent metabolic reactions will not proceed only by the upstream gene, so that metabolic intermediates such as β-carotene accumulate, and a sufficient production amount is not obtained. In other words, it is important to increase the amount of astaxanthin in a total amount of carotenoids including lycopene, β-carotene, echinenone, β-cryptoxanthin, 3′-hydroxyechinenone, zeaxanthin, 3-hydroxyechinenone, canthaxanthin, phonicoxanthin, 4-ketozeaxanthin which are intermediates of astaxanthin synthesis, and astaxanthin and so on. The present inventors found that a sufficient amount of astaxanthin is synthesized without accumulation of metabolic intermediates in expression cells when a DNA chain selected from the group consisting of the above (a), (b), (c), (d), (e) and (f) is introduced rather than using IPP isomerase, for sufficiently producing astaxanthin without stopping at metabolic intermediates of astaxanthin. The present inventors also considered that introduction of DNA chain of (f) is effective for increasing carotenoid. In other words, based on a comparable idea to that for increase in expression amount by introduction of IPP isomerase, the present inventors found that by introducing a DNA chain encoding geranylgeranyl diphosphate synthase which is considered as an upstream synthase according to metabolic concept, an expression amount of geranylgeranyl diphosphate synthase increases, and geranylgeranyl diphosphate which is a product is supplied to the to the carotenoid metabolic system, and thus a total carotenoid amount is increased by a series of carotenoid synthases. Accordingly, combination of a DNA chain selected from (a), (b), (c), (d) and (e) and a DNA chain of (f) dramatically increases a synthesis amount of astaxanthin. Furthermore, the present inventors found that combination of DNA chains of (a), (b), (c), (d), (e) and (f) enables selective synthesis of desired carotenoid. For example, for selective synthesis of β-carotene, (c), (d) and (e) may be combined, and (f) may be further combined for increasing the production amount. For selective synthesis of zeaxanthin, (b), (c), (d) and (e) may be combined, and (f) may be further combined for increasing the production amount. For selective synthesis of lycopene, (d) and (e) may be combined, and (f) may be further combined for increasing the production amount. If a combination of (a) and (b) is available, oxidation of β-carotene selectively proceeds, which enables astaxanthin to be efficiently and selectively synthesized. In order to further increase the production amount of astaxanthin, combinations of (d), (e) and (f) may be used.
In the present invention, when referring to a combination of DNA chains, the DNA chains may be individually used, however they may be genetically coupled serially. The combination number to be coupled can be selected such that a desired function is maximum in the combination. When DNA chains are individually used, they may be inserted into an appropriate plasmid vector. Plasmid vectors should merely function in a host cell into which the plasmid vector is introduced, and an appropriate plasmid vector may be used singly or plural kinds of plasmid vectors may be used insofar as they are not subjected to compatibility restraint.
In the present invention, the term “carotenoid” includes phytoene, lycopene, β-carotene, zeaxanthin, canthaxanthin, astaxanthin, adonixanthin, cryptoxanthin, echinenone, adonirubin, and combinations thereof. Preferably, it is astaxanthin.
Specifically, the present invention provides a DNA chain having a property of increasing a production amount of carotenoid, in particular, astaxanthin, which is selected from the group consisting of a DNA chain encoding a polypeptide having such an enzymatic activity that converts a methylene group at 4 position in β-ionone ring into a keto group (crtW), a DNA chain encoding a polypeptide having such an enzymatic activity that adds one hydroxyl group to a carbon at 3-position of 4-keto-β-ionone ring and/or at 3-position of β-ionone ring (crtZ), a DNA chain encoding a polypeptide having such an enzymatic activity that converts lycopene into β-carotene (crtY), a DNA chain encoding a polypeptide having such an enzymatic activity that converts phytoene into lycopene (crtI), a DNA chain encoding a polypeptide having prephytoene synthase activity (crtB), and a DNA chain encoding a polypeptide having geranylgeranyl diphosphate synthase activity (crtE), and a method of producing carotenoid which comprises introducing the above DNA chain into a carotenoid producing microorganism and culturing the transformed microorganism in a culture medium to thereby increase a carotenoid content of the culture.
In another aspect of the present invention, the present invention relates to a method of preparing a carotenoid producing cell. The method comprises the steps of: introducing a DNA chain, that encodes an enzyme involved in a series of carotenoid synthesis expressed in a cell, into the cell; and preparing and selecting a cell that produces carotenoid in such an amount that is about 1.1 times to 1,000 times the production level of carotenoid produced by the cell prior to introduction of the DNA chain.
The DNA chain according to the present invention is a DNA chain described in (a), (b), (c), (d), (e) or (f), or a DNA chain that hybridizes therewith under a stringent condition.
The wordings “hybridize under a stringent condition” refers to the case where a probe hybridizes with a target sequence thereof but not with other nucleic acids in a complicated mixture of nucleic acids under a stringent condition. The stringent condition depends on the sequence, and differs depending on the environment. For a longer sequence, specific hybridization is achieved at higher temperature. In general, a highly stringent condition is selected such that it is lower by about 5 to 10° C. than the melting temperature of specific sequence at definite ion strength and pH. A low stringent condition is generally selected such that it is lower by about 15 to 30° C. than the melting temperature. The “melting temperature” is the temperature at which 50% of probes that are complementary to a target nucleic acid in equilibrium at definite ion strength, pH and nucleic acid. Any nucleic acids that do not hybridize with each other under a stringent condition are regarded as being substantially identical if the polypeptides coded by these nucleic acids are substantially identical. This occurs, for example, when copies of nucleic acid are generated by using the maximum codon degeneracy which is acceptable by genetic coding. In such a case, nucleic acids typically hybridize under a moderately stringent hybridization condition.
The wording “substantially identical” is used for sequences or partial sequences exhibiting at least 60%, preferably 80%, more preferably 90% or more nucleotide or amino acid residue identity when two nucleic acids or polypeptides are examined by using one of the later-described sequence comparing algorism or examined by manual alignment or visual check, and aligned to achieve the maximum correspondence in a comparison window. This definition also applies to such a sequence that a complement thereof hybridizes with a test sequence.
For sequence comparison, typically one sequence is prepared as a reference sequence, and a test sequence is compared with the reference sequence. When a sequence comparing algorism is used, a test sequence and a reference sequence are inputted to a computer, and partial coordinates are specified as is necessary, and a parameter of sequence algorism program is designated. Default values of the program may be used, or an alternative parameter may be designated. Then, the sequence comparing algorism calculates a percentage of sequence identity of the test sequence with respect to the reference sequence according to the parameter of the program. The method of sequence alignment for comparison is known in the art. An optimum sequence for comparison may be selected, for example, by local homology algorism described in Smith and Watreman, Adv. Appl. Math., 2:482 (1981), homology alignment algorism described in Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), similarity inquiry method described in Person and Lipman, Proc. Natl. Acd. Sci. USA, 85:2444 (1988), and execution of these algorisms with a computer or execution by manual alignment and visual check.
PILEUP is one example of useful algorisms. PILEUP creates multiple sequences from related sequence group using a continuous pairwise alignment and gives relationship and percentage of sequence identity. PILEUP also plots tree or dendgram representing cluster relationship for use in making alignment. Another example of algorism suited for determining percentage of sequence identity and sequence similarity is BLAST algorism (Altschul et al., J. Mol. Biol., 215: 403-410 (1990)). In this algorism, the word lengths are fixed to 3 for protein and 11 for nucleic acid (3 for the case where the sequence is translated by a total of six reading frames). These lengths are the minimum values that are able to give high word scores to adequately significant ones, and are not too long to pass over short but significant patterns. BLAST algorism also conducts statistical analysis concerning similarity of two sequences.
The polypeptide encoded by the DNA chain of the present invention has an amino acid sequence of a sequence selected essentially from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7. In the present invention, the polypeptide encoded by such a DNA chain may have some modifications such as deletion, substitution and addition at some of amino acids unless the carotenoid amount increasing activity as described above is impaired. This corresponds to the fact that “amino acid sequence has substantially a sequence selected essentially from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6 and 7”. For example, those lacking the first amino acid (Met) of these enzymes are also included in the polypeptides or enzymes with modified amino acid sequence. DNA chains of the present invention encoding the respective polypeptides include not only those having base sequences encoding the amino acid sequences represented by SEQ ID NOs: 2, 3, 4, 5, 6 and 7 but also degenerate isomers which encode the same polypeptide but have different degenerate codons.
One means for acquiring a DNA chain having a base sequence encoding an amino acid sequence of the above protein is to chemically synthesize at least a part of the chain length according to a method of synthesizing nucleic acid synthesis, however, in consideration of multiplicity of the binding amino acids, it would be preferable to prepare a genomic DNA of Paracoccus sp. or the like, fragmenting into random fragments by restriction enzyme treatment using an appropriate restriction enzyme such as Sau3AI, preparing a library of Escherichia coli based on the cosmid method, and using a hybridization method with the use of an appropriate probe, rather than the chemical synthetic method. Furthermore, when an appropriate PCR primer can be manufactured, a desired DNA chain may be amplified by PCR method using the prepared genomic DNA as a template.
A DNA chain may be used without modification for transformation of an appropriate cell, however, it may be inserted into a plasmid vector before use. A plasmid vector can be genetically inserted into an appropriate position of a plasmid vector. The appropriate position may be selected so that regions concerning replication of plasmid vector, desired antibiotic marker and transmissivity are not broken.
In insertion to a plasmid vector, the above DNA chain may be genetically inserted without modification, however, a DNA chain having a promoter activity may be added. The term “promoter” used herein refers to a DNA sequence capable of controlling expression of a protein coding region or functional RNA, and as such, lac promoter, trc promoter and the like that function in Escherichia coli can be exemplified. In the case where the DNA chain is expressed in a marine bacterium, there is no limitation insofar as such DNA chain is a DNA chain including a promoter sequence functioning in cells. Preferably, the promoter is derived from marine bacteria. Preferably, by using a promoter of SEQ ID NO: 19, 20 or 21, it is possible to express an inserted gene encoding a carotenoid synthase. Alternatively, a part of polynucleotide of SEQ ID NO: 19, 20 or 21 may be used. A partial region can be identified by comparison of known promoter sequences. Furthermore, these sequences may have insertion or substitution of a base. Furthermore, mutations may be introduced at random and polynucleotides with improved promoter activity may be used. In general, a coding region of enzyme protein or the like is positioned on the 3′ side of the promoter sequence. Commercially available plasmid vectors can be applicable if they already have a promoter sequence, and the promoter functions in a marine bacterium. Furthermore, the orientation in which a DNA chain is to be inserted may be any orientation insofar as the DNA chain functions.
As the plasmid vector, any vectors can be used insofar as they are present stable and replicable in transformed cells. Furthermore, as the plasmid vector, pUC series, pBR series and the like that are used for transformation of Escherichia coli, as well as shuttle vectors linked with a plasmid vector which is replicable in the objective cells can be exemplified. For the details, see published document (Barbara E. Funnell, PLASMID BIOLOGY, ASM press) In Paracoccus sp. of the present invention, a replicable plasmid vector is not particularly known. When there is no established host vector, a broad-host-range plasmid vector may be used. As such a vector, RK2, R751, RSF1010, R1162, pCU1, R46, pSA, R388, RA1 can be exemplified (Barbara E. Funnell, PLASMID BIOLOGY, ASM press). Furthermore, it may be inserted into an appropriate plasmid vector with the use of a replication region of a broad-host-range vector, and used as a shuttle vector. For example, a shuttle vector can be exemplified, which is prepared by inserting a replication region of RK2 vector into an appropriate position of a pUC series vector, and is able to utilize Escherichia coli. Also pBBR series plasmids can also be exemplified, which have relatively small DNA sizes and are replicable in a broad range of hosts. Examples of the pBBR series plasmids include pBBR122, pBBR1MCS, pBBR1MCS2, pBBR1MCS3, pBBR1MCS4, and pBBR1MCS5 (Non-patent document 5). These plasmid vectors are characterized, for example, by different antibiotic markers, and may be selected for use after evaluation of antibiotic resistance of a transformed cell. Furthermore, a plasmid retained by a cell to be transformed may be used.
By introducing a DNA chain of the present invention as described above, or a DNA chain inserted into an appropriate plasmid vector, into an appropriate carotenoid producing microorganism, it is possible to increase a carotenoid content. In the present invention, the following expression vectors are included: pBBR1MCS2CRT, pBBR1MCS2CRTrv, pBBR1MCS2CRTWZ, pBBR1MCS2CRTWZrv, pBBR1MCS2PcrtE1crtE, pBBR1MCS2PcrtE2crtE, pBBR1MCS2PcrtE1crtECRT, and combinations thereof. These vectors will be defined in Examples given later.
Preferred host cells can be broadly found in fungi and bacteria families, and are biological hosts that proliferate in wide ranges of temperature, pH and solvent resistance. For example, either of bacteria, yeast and filamentous fungi is a suitable host for expression of DNA chain of the present invention. Since mechanisms of transcription and translation of DNA chain, and biosynthesis mechanism of protein are common regardless of supply materials of cells, a functional gene will be expressed regardless of a carbon supply material used for culturing of culture. For large-scale culture of microorganism and functional gene expression, a wide variety of simple or complicated carbohydrates, organic acids and alcohols, and saturated hydrocarbons such as methane can be used, while photosynthetic or chemoautotrophic hosts can utilize carbon dioxide. However, a functional gene may be regulated, suppressed or lowered by a specific culture condition which may include forms and amounts of micronutrients including nitrogen, phosphorus, sulfur, oxygen, carbon or inorganic substances. Furthermore, regulation of functional gene may be achieved by a specific regulator substance which is added to a liquid culture and typically is not considered as a source of nutrient or energy.
Examples of the host include microorganism species such as Aspergillus, Trichoderma, Pichia, Candida, Hansenula, Saccharomyces, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhocdococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Myxococcus, Thiobacillus, Methanobacterium, Paracoccus and Klebsiella. Preferred are bacteria belonging to Paracoccus, which may be conveniently identified by those skilled in the art by taking a DNA sequence encoding 16S rRNA as an index. More preferred is Paracoccus sp. strain MBIC1143 for which carotenoid production amount is reported. A DNA base sequence encoding 16S rRNA of Paracoccus sp. strain MBIC1143 is disclosed to public. See, for example, Accession No. AB008114 of National Center for Biotechnology Information which is official database. A base sequence of DNA chain encoding 16S rRNA of Paracoccus sp. strain MBIC1143 is shown by SEQ ID NO: 24. Further preferred are strain TSN18E7 (see Japanese Patent Laid-Open Publication No. 2005-58216), and strain TSTT001 (see specification of Japanese Patent Application No. 2005-314667) which are mutants derived from Paracoccus sp. strain MBIC1143. It is, in particular, a mutant derivative strain which is free from regulation on various metabolic system concerning cell growth due to intracellular accumulation of carotenoids. In other words, it is a mutant strain for which regulation is cancelled by mutation treatment or the like. In cells, when a specific metabolic product accumulates, feedback inhibition or the like may occur by the metabolic product to stop subsequent synthesis of the metabolic product. The wording “canceling of regulation” means suspension of regulation mechanism in cells. There is an industrial successful case in lysine fermentation of amino acid. The mutation treatment may be carried out using a mutation treatment agent which is well-known in the art such as nitrosoguanidine, ethyl methane sulfonic acid, ultraviolet rays, radiation rays, or the like. The strain for which regulation is cancelled may be a natural mutant. Alternatively, mutants after mutation treatment and natural mutants may be isolated and obtained from a selective medium utilizing a metabolic analogue of carotenoids. The metabolic analogue is a substance that has a similar chemical structure to carotenoid, or a substance that shows a physiologic reaction similar to that of carotenoid in various intracellular reaction systems. Examples of the metabolic analog include β-ionone and α-ionone. Furthermore, a gene expression chip of host cell may be created, and an expression profile may be precisely analyzed under various culture conditions, and based on the gene expression profile under the environment where carotenoid accumulates in high density, a gene knockout or knock-in strain may be created and used.
The following is description of outline of a gene transduction procedure to a desired microorganism. A procedure or method for introduction and expression of an exogenous gene into a microorganism such as Escherichia coli includes those commonly used in the field of genetic engineering as well as those shown below in the present invention, and may be practiced according to such a procedure and method (for example, “Vectors for cloning genes”, Method in Enzymology, 216, p. 469-631, 1992, Academic Press and, “Other bacterial systems”, Method in Enzymology, 204, p. 305-636, 1991, Academic Press). Concrete examples include a heat-shock method, and an electroporation method.
As to a method of gene transduction into Paracoccus sp. in the present invention, no established technique is known. In such a case, as a method of introducing a gene in a mild condition, a conjugational transfer method using Escherichia coli is exemplified. The conjugational transfer method is a method in which a plasmid is introduced from a donor bacterium to a recipient bacterium by conjugation between these bacteria, and is advantageous in that the damage to the recipient bacterium is small. The conjugational transfer method using Escherichia coli is classified into two methods: a biparental transfer method and a triparental transfer method. In the former biparental transfer method, by co-culture of a plasmid donor bacterium which is Escherichia coli strain S17-1 incorporating in its chromosome a tra region which is responsible for self-transferability, with a recipient bacterium, it is possible to introduce the plasmid to the recipient bacterium from the donor bacterium by the action of tra (Non-patent document 6) Furthermore, when a plasmid vector (for example, the above pBBR1MCS) having a mob gene (Non-patent document 7) is used, it is possible to effectively introduce the plasmid into the recipient bacterium. The triparental transfer method is a method in which conjugation is caused by mixing Escherichia coli having a helper plasmid (for example, RK1), Escherichia coli having an appropriate plasmid vector and a bacterium to which a gene is to be introduced.
Both methods involve incubation on a filter for a certain time after mixing cells in a medium such as buffer at a certain temperature on a membrane disc. Regarding the conjugation condition for these cases, the temperature is usually, but not limited to, 20 to 30° C., and preferably 25° C. The incubation time is generally from several hours to several days. The mixing ratio of Escherichia coli and a bacterium to which a gene is to be introduced is not particularly limited. This is because when Escherichia coli or yeast to be conjugated is present even in a trace amount, a conjugant is obtained, and such a conjugant can be proliferated through isolation and culture. In order to achieve effective conjugation, the ratio of Escherichia coli and a bacterium to which a gene is to be introduced is, for example, 1:1 or 0.1:1.
After conjugational transfer, the conjugational-transferred cell may be isolated from other cells based on its characteristic acquired as a result of the conjugational transfer. For example, using an antibiotic resistance introduced into the used plasmid vector, only a conjugational-transferred cell into which the plasmid vector is introduced can be proliferated and isolated. All of these methods are well known by those skilled in the art. Furthermore, through combinational use of an antibiotic that prevents growth of Escherichia coli which is a donor of conjugational transfer, more efficient selection of conjugational-transferred cell is realized. Examples of the antibiotic include Carbenicillin, Ampicillin, Cefazollin, Piperacillin, Fosfomycin, Gentamicin, Streptomycin, Neomycin, Amikacin, Tetracyclin, Erythromycin, Lincomycin, Rifampicin, Nalidixic acid, and Novobiocin. Isolation of conjugational-transferred cell can be checked by plasmid extraction, PCR and the like techniques after culturing in an appropriate liquid culture.
By introduction and expression of a group of carotenoid synthesis gene according to the procedure or method of gene transduction to microorganism as described above, it is possible to obtain a microorganism capable of producing a great amount of carotenoid.
Using these transformants and an appropriate culture medium, it is possible to make various carotenoids accumulate in cells. For collection of carotenoids, carotenoid accumulating microorganism may be collected from the culture medium and extracted from an appropriate organic solvent. As an organic solvent, methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethylformamide, and dimethylsulfoxide are exemplified, with acetone being preferred. Furthermore, using a liquid chromatography, high purity separation is realized. As the isolation principle of the liquid chromatography, ion exchange, hydrophobic interaction, molecular sieve and the like can be exemplified. Reverse-phase chromatography and normal-phase chromatography are preferred.
The following examples are intended for concrete explanation of the present invention and not for limitation of the present invention. The gene recombinant experiments made herein are based on a standard method (Sambrook, J., Fritch, E. F., Maniatis, T., “Molecular cloning 3rd edition”, Cold Spring Harbor Laboratory Press) unless otherwise specified.
Example 1
Preparation of Genomic DNA Derived from Paracoccus Strain MBIC1143 and Cloning of Carotenoid Synthesis Gene
Paracoccus sp. strain MBIC1143 was cultured in an OEG culture medium (2 g/L of trypticase peptone, 1 g/L of yeast extract, 8.8 g/L of NaCl; 0.73 g/L magnesium sulfate heptahydrate, 3.6 g/L of anhydrous dipotassium phosphate, 1.4 g/L of potassium phosphate, 1 g/L of D-glucose) at 25° C. (rotary shaking at 120 rpm) for 3 days. Paracoccus sp. strain MBIC1143 was provided by Marin Biotechnology provided by Institute Co., Ltd.
A genomic DNA was prepared using a kit available from Gentra Systems, Inc. (Puregen Genomic DNA isolation kit) (about 50 ng/ml) Using the prepared DNA as a template, a carotenoid synthesis gene was amplified by PCR. Base sequences of genes (crtW, crtZ, crtY, crtI, crtB, crtE) constituting a group of carotenoid synthesis genes of Paracoccus sp. strain MBIC1143 are described in Non-patent document 2 and Patent document 3. Referring to these published data, we created a base sequence containing the above genes. SEQ ID NO: 1 shows a sequence of 7,029 bases. FIG. 1 shows a structure of gene cluster. PCR primers (SEQ ID NO: 13: 5′-gcggatccggcgaccttgcggcgctg-3′) and SEQ ID NO: 14: 5′-cgggatcctgtcgcggtccctgggg-3′) were created with reference to Non-patent document 2. PCR was conducted in the following manner. To 1.0 μL of prepared DNA, 13.5 μL of water, and 25 μL 2×High GC buffer (TAKARA BIO INC.) were added, and heated for 10 minutes at 94° C. After cooling on ice, 8 μL of dNTP, 1.0 μL of 10 pmol/μL forward primer represented by SEQ ID NO: 8, and 1.0 μL of 10 pmol/μL reverse primer represented by SEQ ID NO: 9 were added, and finally 0.5 μL of exTaqDNA polymerase (TAKARA BIO INC.) was added. The reaction included 30 cycles (each cycle consists of a step of 30 sec. at 94° C., a second step of 30 sec. at 60° C. and a third step of 4 min. at 72° C.), followed by reaction for 10 minutes at 72° C. The resultant PCR product was treated with phenol/chloroform, and then subjected to 0.9% agarose electrophoresis to extract and purify an objective product (about 5.4 k base) (QIAgen Gel Extraction Kit available from QIAGEN). The base sequence is shown by SEQ ID NO: 8. The purified DNA (to which a restrictive site of enzyme BamHI is inserted by PCR primer) was treated with a restriction enzyme BamHI, and purified by phenol/chloroform and ethanol precipitation. Then the DNA treated with the restriction enzyme was ligated to the BamHI site of the pUC19 plasmid vector (TAKARA BIO INC.) and after gene transduction by a heat shock method, Escherichia coli strain JM109 was transformed in a LB (Luria-Bertani) agar culture medium containing 100 μg/ml of carbenicillin.
An arbitrary transformant was cultured in a LB culture medium (37° C., 18 hours), and plasmid was extracted using a plasmid extraction kit (available from QIAGEN). Treatment of plasmid with a restriction enzyme BamHI demonstrated the presence of the intended insert. The plasmid vector where a carotenoid synthesis gene from Paracoccus was cloned was named pUCCRT. The structure of the prepared plasmid is shown in FIG. 3. According a method of Japanese patent application No. 2005-106045 as recited above, pUCCRT was checked for exhibition of carotenoid synthesis activity in Escherichia coli. 
Example 2
Preparation of Paracoccus Expression Vector
Plasmid vector pUCCRT was treated with a restriction enzyme BamHI, and a carotenoid synthesis gene fragment (about 5.4 k base) was obtained. Then the fragment was inserted into BamHI site in a broad-host-range vector pBBR1MCS2. Gene transduction into Escherichia coli strain JM109 was conducted by a heat shock method, and then transformation in a LB agar culture medium containing 50 μg/ml of kanamycin was conducted. An arbitrary transformant having acquired kanamycin resistance was cultured in a LB culture medium (37° C., 18 hours), and plasmid was extracted using a plasmid extraction kit (available from QIAGEN). Treatment of plasmid with a restriction enzyme BamHI demonstrated the presence of the intended insert. There are two orientations for insertion of the insert fragment. To be more specific, a vector in which transcription directions of lac promoter and insert fragment in the pBBR1MCS2 vector are the same (pBBR1MCS2CRT) and a vector in which transcription directions of lac promoter and insert fragment in the pBBR1MCS2 vector are opposite (pBBR1MCS2CRTrv). Structures of prepared vectors are shown in FIG. 4.
Example 3
Homogeneous Expression of Carotenoid Synthesis Gene in Paracoccus Bacteria
Each of the vectors pBBR1MCS2CRT and pBBR1MCS2CRTrv into which a carotenoid synthesis gene fragment was cloned was introduced into Escherichia coli strain S17-1 by a heat shock method, and transformation was effected in a LB agar culture medium containing 50 μg/ml of kanamycin and 10 μg/ml of streptomycin. An arbitrary transformant having acquired kanamycin resistance was cultured in a LB culture medium (37° C., 18 hours), and a plasmid was extracted using a plasmid extraction kit (available from QIAGEN) to check whether an objective plasmid was introduced. Then by treatment with a restriction enzyme BamHI, whether the plasmid was properly replicated in Escherichia coli strain S17-1 was checked. The two different plasmid vectors were respectively replicated without occurrence of recombination in Escherichia coli strain S17-1.
Escherichia coli strain S17-1 having the pBBR1MCS2CRT plasmid vector into which a carotenoid synthesis gene is inserted was cultured in a LB culture medium containing 50 μg/ml of kanamycin and 10 μg/ml of streptomycin (37° C.), and a liquid culture containing cells in logarithmic growth phase was obtained. Turbidity (OD 660 nm) was measured, and the culture was diluted in the same culture medium so that the turbidity was 0.1. In parallel with this, Paracoccus bacterium was cultured in an OEG culture medium of Example 1 (25° C.), and a liquid culture containing cells in logarithmic growth phase was obtained. In the same manner as described above, turbidity was measured and the culture was diluted in an OEG culture medium so that the turbidity was 1.0. Each 1.0 mL of these solutions was put into 5 mL-capacity syringe, and the solution in the syringe was passed through a membrane attached to a membrane holder (available from Advantec) to collect cells. After collection of cells, the membrane was removed from the holder, and placed on an OEG agar culture medium (the cells upside), and culturing for causing conjugational transfer was conducted (25° C., 4 hours). After culturing, the membrane was put into 1.0 ml of an OEG culture medium, and conjugational-transferred cells were removed from the membrane filter by agitation. The solution was then diluted in an OEG culture medium to an appropriate concentration, and spread on an OEG agar medium containing 50 μg/ml of kanamycin and 15 μg/ml of amikacin (available from Sigma) and cultured at 25° C. The amikacin was added so as to suppress of growth Escherichia coli. 
A colony appearing at 3 days of culturing was picked up, and cultured in an OEG culture medium containing 50 μg/ml of kanamycin (25° C.) to extract a plasmid. Plasmid extraction was carried out with the use of a plasmid extraction kit available from QIAGEN as is the case with the Escherichia coli strain JM109. Electrophoresis in 0.9% agarose of the resultant liquid extract resulted in the same result as that of pBBR1MCS2CRT carried in Escherichia coli strain S17-1. This demonstrated that plasmid transfer from Escherichia coli to Paracoccus bacterium proceeded successfully. The same examination was conducted for pBBR1MCS2CRTrv.
Example 4
Quantification of Astaxanthin Production in Transformed Paracoccus Bacterium
Paracoccus bacteria having either of plasmids pBBR1MCS2CRT and pBBR1MCS2CRTrv were respectively cultured in an OEG culture medium containing 100 μg/ml of kanamycin (25° C.). Culture was executed in 60 mL of culture medium put into a 100 mL-capacity Erlenmeyer flask equipped with a baffle placed on a rotary shaking incubator rotating at 120 rpm.
Sampling was made at an appropriate point of time, and after cell collection by centrifugal operation, carotenoids was extracted from acetone and quantified. Quantity of carotenoids was measured by high performance liquid chromatography (HPLC) using a reverse-phase column, and conducted in the following operation procedure. To be more specific, a part of liquid culture was centrifuged to collect cells, and the cells were added with an appropriate amount of pure water and suspended for 10 minutes by a tube mixer. Then 9 times amount of acetone to pure water was added and stirred for 30 minutes by a tube mixer. After that, centrifugation at 14,000 rpm for 5 minutes was conducted, and the supernatant was subjected to quantification by HPLC. As the HPLC column, TSKgel ODS-80 (available from TOSOH Corporation) was used, and measurement was conducted at flow rate of 1.0 ml/min and detection wavelength of 470 nm. Using standard astaxanthin (available from Sigma), a calibration curve was prepared, and a production amount of astaxanthin in the culture was calculated. As a control of plasmid introduced strains, a strain having only pBBR1MCS2 vector to which no carotenoid synthesis gene is inserted was also prepared. As shown in Table 1, significant increase in production amount of astaxanthin was observed in the Paracoccus bacteria having a carotenoid synthesis gene.
Furthermore, a wild type strain of Paracoccus sp., strain MBIC1143 was subjected to mutagenesis, and gene transduction into strain TSN18E7 with improved astaxanthin synthesis amount was conducted in a similar manner by conjugational transfer. As is the case with the strain MBIC1143, significant increase in production amount of astaxanthin was observed (Table 1).
In the gene-transduced mutant strain TSN18E7, in particular, the increase is significant at 72 hours of culture. The ability to synthesize astaxanthin which is the final product of carotenoid synthesis in short time demonstrates the effect of plasmid vector that is prepared by insertion of a carotenoid synthesis gene. Paracoccus sp. strain TSN18E7 is deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the number of FERM P-19746.







TABLE 1







Production amount of astaxanthin by gene transduced Paracoccus


bacterium









Production amount of



astaxanthin (mg/L)










Strain
Introduced plasmid
72-hour culture
144-hour culture













Wild-type
pBBR1MCS2
2.0
1.7



pBBR1MCS2CRT
3.6
3.0



pBBR1MCS2CRTrv
3.8
2.9


Mutant
pBBR1MCS2
5.1
20.5


TSN18E7
pBBR1MCS2CRT
30.0
28.1



pBBR1MCS2CRTrv
30.0
28.3









Example 5
Analysis of Promoter Sequence of Inserted Carotenoid Synthesis Gene
Similarly to Example 4, a carotenoid synthesis gene fragment was inserted into vector pBBR1MCS2, and homogenously expressed in a Paracoccus bacterium. Significant increase in production amount of astaxanthin which is one kind of carotenoids was observed. In addition, improvement in activity was observed regardless of the orientation of insertion into a vector. This suggests that the carotenoid synthesis gene is expressed without using the function of lac promoter inserted into the vector pBBR1MCS2. In view of this, we made promoter analysis for a sequence located on upstream side in the transcription direction of the amplified carotenoid synthesis gene (from 1 to 450 described in SEQ ID NO: 1). For analysis, commercially available software GENETYX (available from GENETICS) was used. As a result of analysis, a sequence functioning as a promoter was found on upstream side of crtW gene in the amplified DNA (Table 2). The base sequence from the first position to just proximal to crtW gene in SEQ ID NO: 19 can be estimated as a base sequence having a promoter activity in Paracoccus sp. The promoter score value in Table is a value calculated by the GENETYX software, and it can be interpreted that the higher the value the more likely the promoter activity be present.







TABLE 2







Analyzed promoter sequence










Promoter sequence










Promoter score
−35 region
−10 region





44.97
TGGAAA
TAGTCT


31.95
ATGGAA
TAGTCT









Example 6
Preparation of β-Carotene Oxidase Expression Vector
Genomic DNA of Paracoccus sp. was prepared in a similar manner as described in Example 1. Then, a region containing crtW and crtZ which are genes of β-carotene oxidase was amplified by PCR. The base sequence is shown in SEQ ID NO: 9. For PCR, primers of SEQ ID NO: 13 and SEQ ID NO: 15(′5-cgggatccgcagggcgatcagcccgttggcaagg-3′) were used. Then, to 1.0 μL of the prepared DNA serving as a template, 16.5 μL of water, and 25 μL of 2×High GC buffer (TAKARA BIO INC.) were added and heated for 10 minutes at 94° C. After cooling on ice, 5 μL of dNTP, 2.0 μL of 10 pmol/μL forward primer represented by SEQ ID NO: 13, 2.0 μL of 10 pmol/μL reverse primer represented by SEQ ID NO: 15, and finally 0.5 μL of exTaq DNA polymerase (TAKARA BIO INC.) were added. The reaction included 30 cycles (each cycle consists of a step of 30 sec. at 94° C., a second step of 30 sec. at 60° C. and a third step of 2 min. at 72° C.), followed by reaction for 7 minutes at 72° C. The amplified fragment was examined by agarose electrophoresis and extracted and purified (QIAgen Gel Extraction Kit available from QIAGEN). For allowing insertion into BamHI site of plasmid vector pBBR1MCS2, the purified DNA was digested with a restriction enzyme BamHI. After ligation, gene transduction into Escherichia coli strain JM109 was effected by a heat shock method, followed by transformation in a LB agar culture medium containing 50 μg/ml of kanamycin. An arbitrary transformant having acquired kanamycin resistance was cultured in a LB culture medium (37° C., 18 hours), and plasmid was extracted using a plasmid extraction kit (available from QIAGEN). Treatment of plasmid with a restriction enzyme BamHI demonstrated the presence of the intended insert. There are two orientations for insertion of the insert fragment. To be more specific, a vector in which transcription directions of lac promoter and insert fragment in the pBBR1MCS2 vector are the same (pBBR1MCS2CRTWZ) and a vector in which transcription directions of lac promoter and insert fragment in the pBBR1MCS2 vector are opposite (pBBR1MCS2CRTWZrv). Structures of the prepared vectors are shown in FIG. 5.
Example 7
Expression of β-Carotene Oxidizing Enzyme Expression Vector in Paracoccus sp.
Likewise the above Example 3, each of pBBR1MCS2CRTWZ and pBBR1MCS2CRTWZrv was introduced into Escherichia coli strain S17-1, and a mutant of Paracoccus sp. was transformed by conjugational transfer. After culturing for 3 days, carotenoid was quantified by HPLC. The result is shown in Table 3. In this table, “Ax” represents astaxanthin, and “TC” represents total carotenoid.







TABLE 3







Production amount of carotenoid by gene transduced Paracoccus


bacterium















TC


Strain
Introduced construct
OD660 nm
Ax (mg/L)
(mg/L)














TSN18E7
pBBR1MCS2
4.3
6.1
29.0



pBBR1MCS2CRTWZ
4.1
22.4
27.0



pBBR1MCS2CRTWZrv
3.8
19.3
25.0









As shown in Table 3, synthesis of astaxanthin was significantly increased due to the effect of introduced gene construct, namely increase in expression amount of β-carotene oxidizing enzyme while no increasing effect for total carotenoid was observed.
Example 8
Preparation of Geranylgeranyl Diphosphate Synthesis Gene Expression Vector
Genomic DNA of Paracoccus sp. was prepared in a similar manner to Example 1. Then geranylgeranyl diphosphate synthase gene (crtE) region was amplified by PCR. Since a promoter region which is expected to be present on upstream side of the crtE gene is unknown, referring to the base sequence of SEQ ID NO: 1, we designed and used two sets of PCR primers having different hybridizing regions: a set of primers represented by SEQ ID NO: 16 (5′-ctagtctagatgcttgacaatccgggtgacgcgg-3′) and SEQ ID NO: 17 (5′-tgggagctcatcacgcctaggcgcgcgcggcgtag-3′) and a set of primers represented by SEQ ID NO: 18 (5′-ctagtctagagccggtccactgaccttgttggac-3′) and SEQ ID NO: 17. The former set amplifies a region of about 1.2 k base, and the latter set amplifies a region of 1.1 k base. The respective base sequences are shown by SEQ ID NO: 10 and SEQ ID NO: 11. The longer amplified region was named PcrtE1crtE, and the shorter amplified region was named PcrtE2crtE. As to PcrtE1crtE, first, 1.0 μL of prepared DNA which serves as a template of PCR was added with 16.5 μL of water, and 25 μL of 2×High GC buffer (TAKARA BIO INC.), and heated for 10 minutes at 94° C. After cooling on ice, 5 μL of dNTP, 2.0 μL of 10 pmol/μL forward primer represented by SEQ ID NO: 16, 2.0 μL of 10 pmol/μL reverse primer represented by SEQ ID NO: 17, and finally 0.5 μL of exTaq DNA polymerase (TAKARA BIO INC.) were added. The reaction included 30 cycles (each cycle consists of a step of 30 sec. at 94° C., a second step of 30 sec. at 60° C. and a third step of 2 min. at 72° C.), followed by reaction for 7 minutes at 72° C. The amplified fragment was examined by agarose electrophoresis and extracted and purified (QIAgen Gel Extraction Kit available from QIAGEN). Furthermore, terminals for insertion into a plasmid vector were arranged by digestion with restriction enzymes Xba I and SacI. In a similar manner, PcrtE2 was prepared using primers described in SEQ ID NO: 17 and SEQ ID NO: 18.
Next, pBBR1MCS2 which is a plasmid vector into which an insert is to be inserted was digested with restriction enzymes BtsI and Bsu36I to cause unneeded DNA chains drop out. Following extraction from phenol/chloroform, ethanol precipitation was conducted for purification. Furthermore, single-stranded oligonucleotides described in SEQ ID NOs: 22 (5′-tcatctagaggtaccatatgaagcttgagctcct-3′) and 23 (5′-gagctcaagcttcatatggtacctctaga-3′) were caused to anneal, and the resultant duplex was ligated to the purified DNA fragment. This duplex was designed to include restriction sites of restriction enzymes SacI and XbaI. After ligation, Escherichia coli JM109 was transformed in a LB agar culture medium containing 50 μg/ml of kanamycin, to obtain a vector into which the duplex was inserted. This was named pBBR1MCS2oligo.
Then pBBR1MCS2oligo was digested with restriction enzymes SacI and XbaI to arrange terminals for insertion of the insert. After ligation, Escherichia coli JM109 was transformed in a LB agar culture medium containing 50 μg/ml of kanamycin. An arbitrary colony was picked up and cultured, and a plasmid was prepared. The plasmid was subjected to electrophoresis to confirm that it was an intended construct. The construct having an insert of longer region was named pBBR1MCS2PcrtE1crtE, and the construct having an insert of shorter region was named pBBR1MCS2PcrtE2crtE. The respective structures are shown in FIG. 6.
Example 9
Expression of Geranylgeranyl Diphosphate Synthase in Paracoccus sp. Bacterium
Likewise the above Example 3, each of pBBR1MCS2PcrtE1crtE and pBBR1MCS2PcrtE2crtE was introduced into Escherichia coli strain S17-1, and a mutant of Paracoccus sp. was transformed by conjugational transfer. After culturing for 5 days likewise Example 4, carotenoid was quantified. The result is shown in Table 4.







TABLE 4







Production amount of carotenoid by gene transduced Paracoccus


bacterium










Strain
Introduced construct
OD660 nm
Carotenoid (mg/L)













TSN18E7
—
6.2
34.2



pBBR1MCS2PcrtE1crtE
1.0
Trace amount



pBBR1MCS2PcrtE2crtE
1.1
Trace amount


TSTT001
—
11
30.0



pBBR1MCS2PcrtE1crtE
6.6
44.4



pBBR1MCS2PcrtE2crtE
7.2
43.0









As shown in Table 4, in the mutants TSN18E7 into which these gene constructs were introduced, bacteriolysis was observed, and increase in carotenoid synthesis amount was not observed. On the other hand, in the mutant TSTT001 with improved growth ability, a supply amount of geranylgeranyl diphosphate which is a product was increased by the effect of these constructs, namely increased amount of expression of geranylgeranyl diphosphate synthase, and a carotenoid synthesis amount was increased by the series of carotenoid synthases encoded by chromosome of Paracoccus. The mutant strain TSTT001 is deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the number of FERM P-20670.
Example 10
Promoter Sequence on Upstream Side of Geranylgeranyl Diphosphate Synthesis Gene
As described in Example 7, it was found that carotenoid synthesis amount was significantly increased when an upstream sequence is added to the region of 882 base encoding geranylgeranyl diphosphate synthase. In other words, it was found that a base sequence of about 300 bases or about 200 bases located on upstream side of ORF of geranylgeranyl diphosphate synthase is a DNA chain having a promoter activity. These base sequences are described in SEQ ID NO: 20 and SEQ ID NO: 21.
Example 11
Preparation of Plasmid Vector Combining Geranylgeranyl Diphosphate Synthesis Gene and Carotenoid Synthesis Gene
The plasmid pBBR1MCS2PcrtE1crtE described in Example 7 was digested with a restriction enzyme XbaI. The cut terminal was blunted with a DNA Blunting Kit (TAKARA BIO INC.), and phenol/chloroform extraction, and purification by ethanol precipitation were carried out. Then, the carotenoid synthesis gene fragment that was prepared in Example 2 and digested with a restriction enzyme BamHI was similarly blunted, followed by phenol/chloroform extraction and purification by ethanol precipitation. These are then ligated, and Escherichia coli JM109 was transformed in a LB agar culture medium containing 50 μg/ml of kanamycin. An arbitrary colony was picked up and cultured, and a plasmid was prepared. The plasmid was subjected to electrophoresis to confirm that it was an intended construct. The base sequence of the prepared insert fragment is shown in SEQ ID NO: 12. This construct was named pBBR1MCS2PcrtE1crtECRT. FIG. 7 shows the structure thereof.
Example 12
Expression of Plasmid Vector Combining Geranylgeranyl Diphosphate Synthesis Gene and Carotenoid Synthesis Gene
Likewise the above Example 3, pBBR1MCS2PcrtE1crtECRT was introduced into Escherichia coli strain S17-1, and a mutant of Paracoccus sp. was transformed by conjugational transfer. Likewise Example 4, carotenoid was quantified by HPLC after culturing for 5 days. The result of quantification is shown in Table 5. In this table, “Ax” represents astaxanthin, and “TC” represents total carotenoid. FIG. 8 shows a HPLC pattern of strain TSTT001 at 3 days of culture.







TABLE 5







Production amount of carotenoid by gene transduced Paracoccus


bacterium













OD660

TC


Strain
Introduced construct
nm
Ax (mg/L)
(mg/L)














TSN18E7
—
6.1
20.3
33.2



pBBR1MCS2PcrtE1crtECRT
5.3
30.5
42.2


TSTT001
—
10.5
17.8
31.1



pBBR1MCS2PcrtE1crtECRT
7.4
33.7
56.5









As shown in Table 5, the production amounts of astaxanthin and carotenoid significantly increased owing to the construct pBBR1MCS2PcrtE1crtECRT. That is, due to the expression of geranylgeranyl diphosphate synthase, geranylgeranyl diphosphate which is a synthesis material of carotenoid is synthesized at high density, and then a series of carotenoid synthases are excessively expressed due to the transduced construct to lead efficient synthesis of astaxanthin. As can be seen from FIG. 8, at 3 days of culture, the synthesis amount of astaxanthin was significantly increased due to pBBR1MCS2PcrtE1crtECRT which is a gene introduced into the strain TSTT001.
INDUSTRIAL APPLICABILITY
In the present invention, we revealed the function of gene or gene group that increases carotenoid production amount, and succeeded in improving natural microorganisms having carotenoid productivity. Therefore, by using the microorganisms improved by the present invention, it is possible to dramatically increase the productivity of carotenoids which are useful as feed or food.
BRIEF EXPLANATION OF DRAWINGS
FIG. 1 A chart of carotenoid biosynthesis pathway.
FIG. 2 A view showing a carotenoid synthesis gene.
FIG. 3 A view showing a structure of plasmid vector pUCCRT.
FIG. 4 A view showing structures of plasmid vectors pBBR1MCS2CRT and pBBR1MCS2CRTrv.
FIG. 5 A view showing structures of plasmid vectors pBBR1MCS2CRTWZ and pBBR1MCS2CRTWZrv.
FIG. 6 A view showing structures of plasmid vectors pBBR1MCS2PcrtE1crtE and pBBR1MCS2PcrtE2crtE.
FIG. 7 A view showing structures of a plasmid vector pBBR1MCS2PcrtE1crtECRT.
FIG. 8 A view illustrating the effect of increasing carotenoid synthesis amount of a bacterium recombined by the plasmid vector pBBR1MCS2PcrtE1crtECRT.

## Claims

1. A method of preparing a carotenoid comprising the steps of:
culturing a cell transformed with a DNA chain having a DNA sequence selected from the group consisting of the following (a) to (f) and having a DNA sequence described in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 or a cell transformed with a vector having a DNA sequence selected from the group consisting of the following (a) to (f) in an appropriate culture condition and having a DNA sequence described in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and
isolating the carotenoid from the cell or a culture medium:
(a) a DNA sequence encoding a polypeptide having an enzymatic activity of an enzyme that is found in Paracoccus sp. strain MBIC1143 and converts a methylene group at 4 position in β-ionone ring into a keto group (crtW), described in SEQ ID NO: 2;
(b) a DNA sequence encoding a polypeptide having an enzymatic activity of an enzyme that is found in Paracoccus sp. strain MBIC1143 and adds one hydroxyl group to a carbon at 3-position of 4-keto-β-ionone ring and/or at 3-position of β-ionone ring (crtZ), described in SEQ ID NO: 3;
(c) a DNA sequence encoding a polypeptide having an enzymatic activity of an enzyme that is found in Paracoccus sp. strain MBIC1143 and converts lycopene into β-carotene (crtY), described in SEQ ID NO: 4;
(d) a DNA sequence encoding a polypeptide having an enzymatic activity of an enzyme that is found in Paracoccus sp. strain MBIC1143 and converts phytoene into lycopene (crtI), described in SEQ ID NO: 5;
(e) a DNA sequence encoding a polypeptide having an enzymatic activity of prephytoene synthase that is found in Paracoccus sp. strain MBIC1143 (crtB), as described in SEQ ID NO: 6; and
(f) a DNA sequence encoding a polypeptide having an enzymatic activity of geranylgeranyl diphosphate synthase that is found in Paracoccus sp. strain MBIC1143 (crtE), as described in SEQ ID NO: 7.

2. A DNA chain having a promoter activity in a marine bacterium, described in SEQ ID NO: 20.

3. A DNA chain having a promoter activity in a marine bacterium, described in SEQ ID NO: 21.

4. A continuous oligonucleotide sequence selected from the group consisting of combinations of the DNA chain according to claim 1 and the DNA chain described in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

5. A plasmid vector comprising the oligonucleotide according to claim 4.

6. A cell transformed with the plasmid vector according to claim 5.

7. A method of producing a carotenoid, comprising:
culturing under a condition that enables expression of a polypeptide encoded by the DNA chain sequence of the plasmid vector according to claim 5; and
isolating the carotenoid from a cell or a culture medium of cell.

8. The method according to claim 7, wherein the cell is a prokaryotic host cell.

9. The method according to claim 7, wherein the cell is a marine bacterium.

10. The method according to claim 7, wherein the cell is a bacterium in which 16S rRNA has a DNA base sequence that has a 97% or higher homology to the DNA base sequence of SEQ ID NO: 24.

11. A method of preparing a desired carotenoid or a carotenoid mixture, comprising:
culturing the cell according to claim 6 under an appropriate culture condition;
isolating the desired carotenoid or a carotenoid mixture from the cell or culture medium; and
separating a single carotenoid from other possibly coexisting carotenoids when such a single carotenoid is desired.

12. A method of preparing a food or feed composition, comprising:
executing the method according to claim 11, and then
adding the carotenoid or carotenoid mixture to food or feed.

13. The method according to claim 9, wherein the marine bacterium is Paracoccus sp.

14. The method according to claim 8, wherein the prokaryotic host cell is Escherichia coli or Bacillus.