JP4205398B2 - Genes useful for the synthesis of monocyclic carotenoids and methods for producing monocyclic carotenoids - Google Patents

Description

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Yokoyama, A. and Miki, W. 1995. Isolation of myxol from a marine bacterium Flavobacterium sp. associated with a marine sponge. Fish. Sci. 61:684-686.
Cunningham, F. X. Jr, Chamovitz, D., Misawa, N., Gantt, E. and Hirschberg, J. 1993. Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene. FEBS Lett. 328: 130-138.
Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., and Miki, W. 1995. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene lebel. J. Bacteriol. 177: 6575-6584.
【００４２】
［実施例２］遺伝子操作技術
プラスミドの精製はQIAprep (Qiagen)を用いて行った。制限酵素処理、ライゲーション反応、形質転換等の通常の遺伝子操作実験は、前述のSambrook ら (1989)のMolecular Cloningに示された方法により行った。
【００４３】
［実施例３］フラボバクテリウム属A99-3株から染色体DNAの調製
フラボバクテリウム属（Flavobacterium sp.）A99-3株（MBIC 03313）を1,000 mlのMarine Broth (MB) 培地（Difco製）で、20℃で3日間培養した。菌体を集菌後、STE緩衝液（100 mM NaCl、10 mM TrisCl、1 mM EDTA、pH8.0）で洗浄し、68℃で15分間熱処理した後、5 mg/mlのリゾチームと100 μg/mlのRNase Aを含むI液（50 mM グルコース、25 mM TrisCl、10 mM EDTA、pH8）に懸濁した。37℃で１時間インキュベートした後、250 μg/mlになるようにproteinase Kを加え、37℃で10分間インキュベートした。さらに、最終濃度が1％になるようにN-Lauroylsarcosine・Naを加え、よく混合した後、37℃で１時間インキュベートした。さらに、フェノール/クロロホルム抽出を数回行った後、２倍量のエタノールをゆっくり加えながら、析出してきた染色体DNAをガラス棒に巻き付け、70％エタノールでリンスした後、2 mlのTE緩衝液に溶解して、染色体DNA調製液とした。
【００４４】
［実施例４］コスミドライブラリーの調製
染色体DNAの調製液からファージ粒子を得るところまでの実験法は、Stratagene社のSuperCos 1 Cosmid Vector Kitの取り扱い説明書に従って行った。すなわち、フラボバクテリウム属A99-3株から調製した染色体DNAを制限酵素Sau3AIで部分分解し、コスミドベクターSuperCos 1のBamHI サイトに連結し、LAMBDA INN (Wako, Osaka, Japan)を用いてファージ粒子にパッケージングした。そして、エルウィニア・ウレドボラ のcrtE、crtB、crtI 遺伝子を含むリコペン産生用プラスミドpACCRT-EIBを保持している大腸菌（E. coli）XL1-Blue MR株に、そのファージを感染させ、ApとCmの両方に耐性のコロニーをApとCmを含むLBプレート上に1,000個得た。
【００４５】
［実施例５］リコペンβ-モノシクラーゼ遺伝子の単離
pACCRT-EIB（培地成分からリコペンを合成できる遺伝子を含む）を保持した大腸菌XL1-Blue MR株に、フラボバクテリウム属A99-3株のリコペンβ-モノシクラーゼ（lycopene β-monocyclase）遺伝子を含むDNA断片がSuperCos 1 ベクターにより導入・発現されていれば、このクローンはリコペンをさらに代謝して、γ-カロチンを作るはずである。リコペンはピンク色を呈するが、γ-カロチンは（オレンジ色を帯びた）黄色を呈するため、プレート上のApとCmの両方に耐性の1,000コロニーから目で見て黄色を帯びたようなコロニーを300個選んだ。色の変化は不明確であったため、これらの大腸菌を液体培養し、菌体をアセトン抽出したものを用いて、フォトダイオードアレー［photodiode array (PDA)］検出を伴った高速液体クロマトグラフィー（HPLC）分析を行った（詳細は実施例８に記載）。HPLCの保持時間（33.1分）と紫外可視吸収スペクトル（λmax、434、465、494 nm）（Jeffrey et al, 1997）により、γ-カロチンを生成していると考えられる３クローンを得た。ちなみに、この条件でのリコペン及びβ-カロチンの保持時間はそれぞれ、23.6分及び46.6分であった。
【００４６】
３クローンの中の1クローンに含まれていたフラボバクテリウム属（Flavobacterium sp.）A99-1株由来の40 kbのDNA断片を含むプラスミドをpSC0601と名付け、その後の解析に用いた。
【００４７】
Jeffrey, S. W., Mantoura, R. F. C., and Wright, S. W. (edited). 1997. Phytoplankton pigments in oceanography: Guidelines to Modern Methods. UNESCO Publishing.
【００４８】
［実施例６］ サブクローニングと塩基配列の決定
プラスミドpSC0601の挿入DNA断片をいくつかの制限酵素による消化後、pBluescript II KS^-（Toyobo）に挿入し、大腸菌JM109を形質転換した。これらの大腸菌を液体培養し、菌体をアセトン抽出したものを用いて、実施例８のHPLC分析により、いくつかのクローンに活性があることを確認した。そのうちの１つである、4.7 kbの SalI-SacI断片がpBluescript II KS^-のSalI-SacI部位に挿入されたプラスミドをpBS0603と名づけ、このDNA断片の塩基配列を決定した。DNA sequencing kit (Dye Terminator Cycle Sequence; Perkin-Elmer) と model 3700 DNA sequencer (Perkin-Elmer) を付属のプロトコールに従って用いDNA断片（4.7 kb）の塩基配列を両方向決定した。決定された塩基配列はSequencher, version 3.0 (Gene Codes Corporation)を用いて解析し、確定したORFのホモロジー検索はBLAST (Altschul and Lipman, 1990)によって行った。
【００４９】
その結果、リコペンβ-モノシクラーゼ遺伝子（crtY- γ）であると考えられる、377個のアミノ酸配列をコードする1134 bpのオープンリーディングフレーム（ORF）が見出された。この遺伝子にコードされる377個のアミノ酸配列は、既存のパラコッカス属（Paracoccus sp.）MBIC01143株（Agrobacterium aurantiacum）やエルウィニア・ウレドボラ（Erwinia uredovora）等のリコペンβ-シクラーゼ（CrtY：リコペンの両端を環化する酵素）（Misawa et al, 1995）とアミノ酸配列全体にわたって相同性を有していたが、相同性の程度は低かった。すなわち、このフラボバクテリウム属（Flavobacterium sp.）A99-1株のリコペンβ-モノシクラーゼ（CrtY-γ）は、パラコッカス属MBIC01143株、及びエルウィニア・ウレドボラのリコペン β-シクラーゼ（CrtY）とそれぞれ、22％及び19％の同一性（identity）を有していた。
【００５０】
更に、上記のリコペンβ-モノシクラーゼ（crtY- γ）遺伝子の隣に、239個のアミノ酸配列をコードする720 bpのオープンリーディングフレーム（ORF）が見出された。この遺伝子にコードされる239個のアミノ酸配列は、既存のロドバクター（Rhodobacter）属細菌等のCrtA［2'-モノオキシゲナーゼ（2'-monooxygenase）］（Armstrong et al, 1989）とアミノ酸配列全体にわたって相同性を有していた。これが、今回、リコペンβ-モノシクラーゼ活性を増強する遺伝子として発見されたものである。
【００５１】
Altschul, S. F. and Lipman, D. J. 1990. Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. USA 87: 5509-5513.
Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., and Miki, W. 1995. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177: 6575-6584.
Armstrong, G. A., Alberti, M., Leach, F., and Hearst, J. E. 1989. Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol. Gen. Genet. 216: 254-268.
【００５２】
［実施例７］ 色素分析用プラスミドの作製
実施例６で得られたプラスミドpBS0603を材料として用いて、色素分析用に次の2種類のプラスミドを作製した。
pBS0603から1.54 kb EcoRV-EcoRI断片を除き、代わりに、EcoRI-NotI-BamHIアダプター（BamHI末端側は平滑）（Takara）を挿入したプラスミドを作製し、pBS0603dDOと名付けた。本プラスミドには、pBluescript II KS^-ベクターに、フラボバクテリウムA99-3株のcrtY- γとcrtAの２遺伝子が挿入されている。次に、リコペンβ-モノシクラーゼ（crtY- γ）遺伝子のみを大腸菌で発現させるためのプラスミド（pG0604）を以下のようにして作製した。オリゴヌクレオチド5' TGTCTGCAGGATTTCAACCGTATTTGATT 3'と5' TATGGATCCCAATTCTTCAATCTTAAAGGAG 3'を用いて、プラスミドpSC0601を鋳型としてcrtY- γ遺伝子をPCRにより増幅し、得られた増幅物をpGEM-T Easyベクター（Promega製）に、このベクターの取り扱い説明書に従ってクローン化して作製した。PCRは、94°Cで10分間の加熱後、94℃で1分、50℃で1分、72℃で2分を繰り返し40回行い、最後に72℃で10分間伸長反応を行った。
【００５３】
［実施例８］ 大腸菌に蓄積されたカロテノイドの抽出と解析
プラスミド pACCRT-EIBを含む大腸菌（E. coli）はリコペンを細胞内に蓄積する。 pACCRT-EIB とSuperCos 1 によるコスミドを含む 大腸菌（E. coli）XL1-Blue MR、またはpACCRT-EIBとpBluescript II KS^-によりサブクローンされたプラスミドを含む大腸菌JM109、またはpACCRT-EIBとpG0604を含む大腸菌JM109は、CmとAp を含むLB 培地で２日間、30℃で振盪培養された（100 rpm）。そして、培養液から菌体を集菌し、アセトンを加え、室温で30分間撹拌した。抽出液を14,000 ｘ gで5 分間（4°C）遠心し, カロテノイド色素抽出液である上澄み液をHPLC（model 2695、Waters製）分析にかけた。条件は、25°C で逆相カラム(3.9 ｘ 300 mm, Nova-pak HR 6 μm C₁₈; Waters製)を用い、アセトニトリル（acetonitrile）−メタノール（methanol）− 2-プロパノール（2-propanol）（90：6：4）で、1 ml/minの速さで展開し、Waters model 2996 PDA 検出器によりモニターした。
【００５４】
プラスミドpBS0603dDO（crtY- γとcrtAを含む）またはpG0604（crtY- γを含む）がpACCRT‐EIBとともに導入された大腸菌を液体培養し、菌体をアセトン抽出したものを用いてHPLC分析を行った。その結果、両者のカロテノイド総量はほぼ同じであったが、前者が85％のγ-カロチンと15％のβ-カロチンを合成していたのに対して、後者は40％のγ-カロチンと60％のβ-カロチンを合成したことが明らかとなった（γ-カロチンの同定は実施例9参照）。なお、両者とも基質のリコペンはほとんど残っていなかった。以上の結果から、CrtY-γはリコペンからγ-カロチンを作るが、培養条件によっては、生成したγ-カロチンの一部をβ-カロチンにまで変換することがわかった。また、CrtAがCrtY-γと共存すると、γ-カロチンの合成量が増えたことから、フラボバクテリウムA99-3株のCrtAはリコペンβ-モノシクラーゼ活性を増強することがわかった。なお、上記のcrtY- γ遺伝子の変わりに、エルウィニア属細菌のcrtY遺伝子をpACCRT-EIBとともに用いると、上記と同等の条件では、ほぼ100％のβ-カロチンが得られ、γ-カロチンは得られない。
【００５５】
［実施例９］ γ-カロチンの同定
プラスミドpBS0603dDOを、pACCRT-EIBを含む大腸菌（E. coli）JM109株に導入し、Ap及びCmを含むLB培地2,000 mlで30℃、２日間培養した（100 rpm）。培養液から集菌した菌体を、300 mlのアセトンにより抽出した。これを濃縮後、200 mlのへキサンにより2回抽出した。さらに、へキサン層を濃縮し、シリカゲルカラムクロマトグラフィーにかけ、へキサン−酢酸エチル（50：1）で展開溶出することにより、γ-カロチンと考えられる純品を1 mg得た。
【００５６】
本物質による紫外-可視吸収スペクトル、FD-MSスペクトル（日立二重収束質量分析計 M-2500）（m/e 536）、及び¹H-NMR（500 MHz、JEOLα）スペクトルにより、本品はγ-カロチン（β,ψ-carotene）と同定された。
【００５７】
【発明の効果】
本発明により、自然界には微量にしか存在しない単環式カロテノイドを大量に生産することが可能になる。
【００５８】
【配列表】

【図面の簡単な説明】
【図１】エルウィニア属細菌およびパラコッカス属細菌における各種カロテノイド合成遺伝子の機能と生合成経路を示す図。
【図２】海洋細菌フラボバクテリウム属（Flavobacterium sp.）A99-3株における各種カロテノイド合成遺伝子の機能と推定されたカロテノイドの生合成経路を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a gene encoding an enzyme responsible for the synthesis of γ (gamma) -carotene (γ-carotene, γ-carotene), which is a key carotenoid for synthesizing various monocyclic carotenoids. The present invention relates to a method for producing various monocyclic carotenoids including γ-carotene using expressed microorganisms.
[0002]
[Prior art]
Carotenoid (also called carotenoid) is a general term for pigments that are abundant in nature and usually consist of an isoprene skeleton with 40 carbon chains. To date, more than 600 carotenoids have been isolated (Pfander, H., ed., Key to Carotenoids, Basel, Birkhauser, 1987). Recently, the carotenoid's preventive effect on various cancers (cancers) has attracted attention, and many reports have been made (for example, Tsubasa Nishino, Noriaki Murakoshi, Masamitsu Yano, Food Style 21, 4, 53-55). Giovannucci, E., Ascherio, A., Rimm, EB, Stampfer, MJ, Colditz, GA, Willet, WC, J. National Cancer Institute, 87, 1767-1776, 1995).
[0003]
Despite the variety of carotenoids, the types of carotenoids used so far in cancer prevention studies (human epidemiological studies, animal administration studies, etc.) have been very limited. These carotenoids are also called β-carotene (also called β-carotene, β-carotene: chemically synthesized product), lycopene (also called lycopene, also called lycopene: extracted from tomato), α-carotene (also called α-carotene, α-carotene) : Extracted from palm oil), lutein (extracted from marigold), astaxanthin (extracted from krill and the like), fucoxanthin (extracted from edible seaweed) and the like. As a result of cancer prevention tests using these pigments, it has been clarified that the cancer prevention effect of carotenoids varies depending on the type of carotenoid. As an example, I would like to show the results of an experiment using mice conducted by Nobuo Takasuka et al. At the National Cancer Center Research Institute (1996 Carotenoid Research Conference Report). The incidence of lung cancer (ddy mouse lung two-stage carcinogenesis model) is 40% for mice receiving lycopene or α-carotene, 70% for mice receiving lutein or astaxanthin, and β-carotene, assuming that control mice not receiving carotenoids are 100% The administered mice had a cancer incidence of 139% (fucoxanthin was not performed). The incidence of liver cancer (mouse natural liver cancer carcinogenesis model) is 30% for mice receiving astaxanthin or fucoxanthin, 50% for mice receiving α-carotene or lutein, and β for control mice not receiving carotenoids. -Carotene administration mice had 70% cancer incidence and lycopene administration mice had 100% cancer incidence. The incidence of skin cancer (mouse skin cancer carcinogenesis model) was 10% for mice treated with fucoxanthin or lycopene, and 100% for mice treated with astaxanthin, assuming that the control mice not receiving carotenoids were 100%. (No other carotenoids are implemented). Comparing the results of these three carcinogenesis models, the effect of lycopene, which was highly effective in suppressing lung cancer and skin cancer, was ineffective in suppressing liver cancer, and the effect of astaxanthin that was highly effective in suppressing liver cancer. It can be seen that there is no effect in suppressing skin cancer.
[0004]
The above results show that, among the more than 600 types of carotenoids, less than 10 types of studies that have been studied for their cancer prevention effects in research above the level actually using animal individuals, Regardless, it shows that carotenoid's individuality is recognized in the cancer prevention effect of carotenoid. It seems that the biggest cause of the small number of carotenoids that have been actually studied is that the types of carotenoids that can be extracted and purified in large quantities are limited to the above.
[0005]
As an effective means for solving the above problem, a method of mass-producing a target carotenoid in yeast or Escherichia coli incorporating a carotenoid biosynthesis gene can be considered. For example, Miura et al. Of Kirin Brewery, Inc., an edible yeast Candida utilis that cannot biosynthesize carotenoids (Candida utilis) Was successfully introduced and expressed as a carotenoid biosynthetic gene group to synthesize astaxanthin, β-carotene, and lycopene by 0.4 to 1.1% (Miura, Y., Kondo, K., Saito, T.). , Shimada, H., Fraser, PD, Misawa, N., Appl. Environ. Microbiol., 64, 1226-1229, 1998). According to this genetic recombination method, it is possible to produce a large amount of carotenoids that have not been recognized in nature until now, or have existed only in trace amounts, by combining various biosynthetic genes. Become. For example, Takaichi et al. Of Nippon Medical School produced parasiloxanthin, which was previously reported only in trace amounts in catfish, as a major carotenoid product in recombinant Escherichia coli (Takaichi, S., Sandmann, G., Schnurr, G., Satomi, Y., Suzuki, A., Misawa, N. Eur. J. Biochem., 241, 291-296, 1996). There is also a report that astaxanthin-β-diglucoside, an “unnatural” carotenoid that has never been reported in nature, was synthesized in recombinant E. coli (Yokoyama, A., Shizuri , Y., Misawa, N., Tetrahed. Lett., 39, 3709-3712, 1998). These examples are carotenoids that are cyclized at both ends (having a β-ionone ring), but there are also examples of acyclic carotenoids that remain linear at both ends. For example, Albrecht et al. At the University of Frankfurt have reported 1,1'-dihydroxy-3,4,3 ', 4'-tetradehydrolycopene (1,1'-dihydroxy), an acyclic carotenoid that has never been reported in nature. -3,4,3 ', 4'-tetradehydrolycopene) is produced in recombinant E. coli (Albrecht, M., Takaichi, S., Steiger, S., Wang, Z. -Y., Sandmann, G. , Nature Biotechnol., 18, 843-846, 2000).
[0006]
The most widely used carotenoid biosynthetic gene for the production of various recombinant microorganisms for carotenoid production is Erwinia (Erwinia) Bacteria (Erwinia Uredobora (Erwinia uredovora) Etc.). Genes obtained from Erwinia bacteriacrtE,crtB,crtI,crtY,crtZ,crtX The functions of biosynthetic enzymes (CrtE, CrtB, CrtI, CrtY, CrtZ, CrtX) encoded by these genes are shown in FIG. 1 (see Non-Patent Document 1). If you want to biosynthesize astaxanthin, you can also use the marine bacterium Paracoccus (Paracoccus) Genus bacteria [Paracoccus sp. MBIC01143 (Agrobacterium aurantiacum) Etc]crtZ WhencrtW Genes are used (Figure 1). Erwinia bacteriacrtE,crtB,crtI,crtYEscherichia coli into which the gene has been introduced and expressed synthesizes β-carotene.crtZ WhencrtWWhen the gene is introduced and expressed, the recombinant Escherichia coli synthesizes astaxanthin. In addition, E. coli that synthesizes this astaxanthin has an Erwinia bacterium.crtXWhen the gene is introduced and expressed, the recombinant E. coli synthesizes “non-natural” astaxanthin-β-diglucoside (FIG. 1).
[0007]
When synthesizing acyclic carotenoids such as 1,1'-dihydroxy-3,4,3 ', 4'-tetradehydrolycopene, it is a cyclase genecrtYRhodobacter (photosynthetic bacterium) for introduction and expression into Escherichia coli that made linear carotenoids such as lycopene without using genesRhodobacter) Genus bacteria (Rhodobacter capsulatus Etc.) derived from a linear carotenoid-modifying enzyme genecrtC(1'-hydrase),crtD(3 ', 4'-desaturase) gene etc. are used.
[0008]
As described above, the use of carotenoid biosynthetic genes for mass production of “non-natural type” carotenoids that are limited or have not been confirmed in nature. Is being shown to be possible. However, all of the carotenoids synthesized to date by this biotechnological method are carotenoids that are cyclized at both ends (for example, astaxanthin-β-diglucoside, parasiloxanetin, etc.), or acyclic that is not cyclized at both ends. It was either a carotenoid (eg, 1,1′-dihydroxy-3,4,3 ′, 4′-tetradehydrolycopene).
[0009]
It is known that γ-carotene, a monocyclic carotenoid with only one cyclized, is contained in palm oil, but the content of γ-carotene is only a few percent of the total carotenoids contained in palm oil. Large-scale preparation was impossible. Palm oil carotene includes 60-70% β-carotene, 30-40% α-carotene, several percent γ-carotene and lycopene. Palm oil carotene has been known to have an excellent preventive effect against several cancers such as lung cancer and liver cancer, and a small amount of γ-carotene is likely to be effective (high Alpha, food additive: palm oil carotene 30% vegetable oil suspension, Lion Corporation brochure; Kyoto Prefectural University of Medicine, Professor Tsubasa Nishino, personal communication). However, monocyclic carotenoids such as γ-carotene exist only in trace amounts in the natural world, including the plant kingdom, and cannot be produced in large quantities. Therefore, various cancer prevention tests can be performed. There wasn't.
[0010]
[Non-Patent Document 1]
Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., Miki, W., J. Bacteriol., 177, 6575-6584 , 1995)
[0011]
[Problems to be solved by the invention]
An object of the present invention is to obtain an efficient method for producing monocyclic carotenoids such as γ-carotene, using an enzyme gene that synthesizes γ-carotene and using a recombinant microorganism into which this gene has been introduced and expressed. Is to provide.
[0012]
[Means for Solving the Problems]
The present inventors are a new species of marine bacterium Flavobacterium belonging to the CFB (Cytophaga-Flavobacterium-Bacterioides) group (Flavobacteriumsp.) The strain A99-3 (MBIC03313) was able to produce the monocyclic carotenoid myxol, and no synthesis of carotenoids with cyclized ends was observed. As a result of intensive research, we succeeded in obtaining a cyclase gene that synthesizes γ-carotene from lycopene from this marine bacterium. It is also necessary to synthesize lycopene from farnesyl pyrophosphate (FPP).crtE,crtB,crtI It was confirmed that the recombinant Escherichia coli can synthesize γ-carotene by further introducing and expressing this gene into E. coli into which the gene has been introduced and expressed. At this time, β-carotene, which is more cyclized than γ-carotene, is also synthesized depending on the culture conditions, but a gene that suppresses this β-carotene synthesis and improves the yield of γ-carotene was also found.
The present invention has been completed based on the above findings.
[0013]
That is, the first of the present invention is a gene encoding a peptide shown in the following (a), (b), or (c).
(A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B) a peptide having an lycopene β-monocyclase activity, comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2;
(C) a peptide derived from a bacterium encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA that hybridizes with the DNA complementary thereto under stringent conditions, and having a lycopene β-monocyclase activity .
[0014]
The second of the present invention is a microorganism obtained by introducing the above gene together with other carotenoid biosynthesis genes.
[0015]
A third aspect of the present invention is a microorganism obtained by introducing the above gene into a microorganism containing another carotenoid biosynthesis gene.
[0016]
A fourth aspect of the present invention is obtained by further introducing a gene encoding a peptide shown in the following (d), (e), or (f) into the above microorganism (the second or third microorganism of the present invention). It is a microorganism.
(D) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4,
(E) a peptide consisting of an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 4 and enhancing lycopene β-monocyclase activity;
(F) a peptide derived from a bacterium that is encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 or a DNA that hybridizes with the DNA complementary thereto under stringent conditions and enhances lycopene β-monocyclase activity peptide.
[0017]
A fifth aspect of the present invention is a method for producing a monocyclic carotenoid, wherein the microorganism is cultured in a medium to obtain a monocyclic carotenoid from the culture or the microbial cell.
[0018]
The sixth of the present invention is a peptide shown in the following (a), (b), or (c). (A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B) a peptide having an lycopene β-monocyclase activity, comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2;
(C) a peptide derived from a bacterium encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA that hybridizes with the DNA complementary thereto under stringent conditions, and having a lycopene β-monocyclase activity .
[0019]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
1. Gene source marine bacterium Flavobacterium genus A99-3
The marine bacterium Flavobacterium genus (the source of the gene of interest)Flavobacteriumsp.) A99-3 strain (MBIC 03313)Homaxinella sp.). The carotenoid produced by this marine bacterium was identified as Mikisol by Yokoyama and the trunk of the Marine Biotechnology Research Institute. This result has been filed on October 15, 1993 as “Mixol production method and its use” (Japanese Patent Laid-Open No. 7-107993). This bacterium has been deposited on October 12, 1993 (deposit number P-13903). This marine bacterium also produces myxolFlavobacterium sp. P99-3 (Yokoyama, A, Miki, W., Fisheries Sci., 61, 684-686, 1995; “P99-3” here is the same strain as A99-3 ).
[0020]
16S rDNA and so on at Marine Biotechnology Research InstitutegyrBWhen the position of the bacterium was classified using a gene, the bacterium was considered to be a new genus and new species within the CFB (Cytophaga-Flavobacterium-Bacterioides) group (unpublished data). The closest genus is dice flexus (a psychrophilic bacterium isolated from Antarctica etc.)Psychroflexus) Genus (unpublished data).
[0021]
2. Estimation of myxol biosynthetic pathway in marine bacterium Flavobacterium genus A99-3
Marine bacterium Flavobacterium (Flavobacteriumsp.) The biosynthesis pathway of mixol estimated in the A99-3 strain is shown in FIG. The biosynthetic pathway to lycopene is thought to be the same as that of Erwinia bacteria. The following cyclase lycopene β-monocyclase (called lycopene β-monocyclase; called CrtY-γ) is unique in this bacterium, and it seems that only one of the lycopene is cyclized to produce γ-carotene. Next, Erwinia bacteriacrtZIt is considered that 3-hydroxy-γ-carotene is produced when a gene corresponding to is activated. Rhodobacter bacteria in γ-carotenecrtC(1'-hydolase; 1'-hydolase) andcrtDWhen the (3'4'-desaturase; 3'4'-desaturase) gene is activated, 3 ', 4'-didehydro-1', 2'-dihydro-β, ψ-carotene-1'-ol (3'4 '-didehydro-1'2'-dihydro-β, ψ-caroten-1'-ol) is considered to be generated. When these three enzymes CrtZ, CrtC, CrtD, 2′-monooxygenase, and the like continuously act on γ-carotene, it is considered that the final product, mixol, is produced.
[0022]
3. Gene encoding cyclase lycopene β-monocyclase (gene of the present invention)
The gene of the present invention encodes a peptide shown in the following (a), (b), or (c). (A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (b) an amino acid sequence comprising one or more amino acids added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 2, and lycopene β-mono A peptide having a cyclase activity, (c) a peptide derived from a bacterium encoded by DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or DNA complementary thereto, A peptide having monocyclase activity.
[0023]
The peptide (a) is a peptide consisting of 377 amino acids having a lycopene β-monocyclase activity obtained from Flavobacterium genus A99-3 (also referred to as CrtY-γ).
[0024]
The peptide of (b) is a peptide in which a mutation that does not cause loss of lycopene β-monocyclase activity is introduced into the peptide of (a). Such mutations include artificial mutations in addition to mutations that occur in nature. Examples of means for causing artificial mutation include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982), but are not limited thereto. The number of mutated amino acids is not limited as long as lycopene β-monocyclase activity is not lost, but is usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids. Yes, most preferably within 5 amino acids.
[0025]
  The peptide of (c) is a peptide having lycopene β-monocyclase activity derived from bacteria obtained by utilizing hybridization between DNAs. The “stringent condition” in the peptide of (c) refers to a condition in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions arePreferably" 0.2 × SSC , 0.1 % SDS , 65 It is about “° C.”.DNA obtained by hybridization usually has high homology with the DNA represented by the nucleotide sequence described in SEQ ID NO: 1. High homology meansPreferably 75 % Homology, more preferably 90 % Homology.
[0026]
The gene of the present invention can be obtained, for example, as follows. First, the marine bacterium Flavobacterium (Flavobacteriumsp.) A99-3 strain cosmid library was prepared and Erwinia bacteria such as Erwinia uredoboracrtE,crtB,crtI Infect the E. coli host (red) into which the gene has been introduced and expressed. After infection, select colonies that show a slightly yellowish red color of Escherichia coli and analyze them by high performance liquid chromatography (HPLC) to find bacteria that produce monocyclic carotenoids of γ-carotene or its metabolites. Thus, a DNA fragment containing the gene of the present invention can be obtained.
[0027]
4). Genes that enhance the activity of lycopene β-monocyclase
When a gene encoding a peptide shown in the following (d), (e), or (f) is introduced into a microorganism together with the lycopene β-monocyclase gene of the present invention and expressed, a monocyclic compound including γ-carotene The yield of carotenoids can be increased. (D) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) an amino acid sequence comprising one or more amino acids added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 4, and lycopene β-mono A peptide that enhances cyclase activity, (f) a peptide derived from a bacterium encoded by a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 or a DNA complementary thereto, -Peptides that enhance monocyclase activity.
[0028]
The peptide (d) is a peptide (referred to as CrtA) having a 239 amino acid sequence that enhances lycopene β-monocyclase activity obtained from Flavobacterium genus A99-3.
[0029]
The peptide of (e) is a peptide in which a mutation is introduced into the peptide of (d) so as not to lose the ability to enhance lycopene β-monocyclase activity. Such mutations include artificial mutations in addition to mutations that occur in nature. The number of mutated amino acids is not limited as long as the ability to enhance lycopene β-monocyclase activity is not lost, but is usually within 20 amino acids, preferably within 12 amino acids, more preferably 6 Within amino acids, most preferably within 3 amino acids.
[0030]
The peptide (f) is a peptide that enhances the lycopene β-monocyclase activity derived from bacteria obtained by utilizing hybridization between DNAs. The “stringent conditions” in the peptide of (f) refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are as described above.
[0031]
The gene can be obtained, for example, as follows. First, the marine bacterium Flavobacterium (Flavobacteriumsp.) A99-3 strain cosmid library was prepared and Erwinia bacteria such as Erwinia uredoboracrtE,crtB,crtI Infect the E. coli host (red) into which the gene has been introduced and expressed. After infection, select colonies that show a slightly yellowish red color of Escherichia coli and analyze them by high performance liquid chromatography (HPLC) to find bacteria that produce monocyclic carotenoids of γ-carotene or its metabolites. As a result, a DNA fragment containing the gene can be obtained.
[0032]
5). Monocyclic carotenoid production process
The method for producing a monocyclic carotenoid of the present invention is characterized in that the gene of the present invention is introduced into a microorganism, and the microorganism is cultured in a medium to obtain a monocyclic carotenoid from the culture or the cells. It is. Here, the monocyclic carotenoid refers to a carotenoid in which only one end is cyclized, for example, γ-carotene, 3-hydroxy-γ-carotene, 3 ′, 4′-didehydro-1 ′, 2 ′. This includes -dihydro-β, ψ-carotene-1′-ol, mixol and the like.
[0033]
In addition to the gene of the present invention, other carotenoid biosynthetic genes are introduced into the microorganism, but this is not necessary when the microorganism originally contains other carotenoid biosynthetic genes. However, when a microorganism having other carotenoid biosynthetic genes originally produces β-carotene or its metabolite, it may be necessary to devise measures such as suppressing the spontaneous lycopene β-cyclase activity.
[0034]
Examples of the microorganism to be used include Escherichia coli, but other microorganisms may be used.
[0035]
Other carotenoid biosynthetic genes include at least the genes required to synthesize lycopene from FPP or geranylgeranyl pyrophosphate (GGPP). Specific examples of such genes include enzyme genes that synthesize GGPP from FPP.crtE, A gene that synthesizes phytoene from two molecules of GGPPcrtBSynthesize lycopene from phytoenecrtI Examples thereof include genes (usually those derived from Erwinia bacteria).
[0036]
If these genes are introduced into an appropriate expression vector and introduced into the microorganism to be expressed, the recombinant microorganism will produce lycopene (the substrate FPP can be produced by all microorganisms. GGPP is also a microorganism. Some of them have a low synthesis amount, but all microorganisms can be made.) To the lycopene-producing microorganism, the gene of the present invention (a gene encoding lycopene β-monocyclase,crtY- γ) Is further introduced and expressed, the microbe will produce γ-carotene.
[0037]
In addition to the above-mentioned recombinant microorganism, a gene that enhances lycopene β-monocyclase activity (crtA), The microorganism will produce more γ-carotene.
[0038]
Furthermore, to such microorganisms,crtZIf the gene is introduced and expressed, the microorganism will make 3-hydroxy-γ-carotene,crtCas well ascrtDOnce the gene is introduced, the microorganism will make 3 ', 4'-didehydro-1', 2'-dihydro-β, ψ-carotene-1'-ol,crtZ,crtC,crtDWhen the 2'-monooxygenase gene (usually derived from Rhodobacter spp.) Is introduced and expressed, the microorganisms will make the final product monocyclic carotenoid, myxol Become.
[0039]
Information on vectors of various microorganisms such as Escherichia coli and yeast and methods for introducing and expressing foreign genes are described in many experimental documents (for example, Sambrook, J., Russel, DW, Molecular Cloning A Laboratory Manual, Three^rd Edition, CSHL Press, 2001), vector selection, gene introduction, and expression can be performed according to them.
[0040]
【Example】
[Example 1] Strains, plasmids, growth conditions
The strains and plasmids used in the present invention are shown in Table 1. Strains were cultured at 30 ° C. using Luria-Bertani (LB) medium (Sambrook et al, 1989). As needed, ampicillin (Ap: 100 μg / ml) or chloramphenicol (Cm: 20 μg / ml) was added to the medium.
[0041]
[Table 1]

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Yokoyama, A. and Miki, W. 1995. Isolation of myxol from a marine bacterium Flavobacterium sp. Associated with a marine sponge. Fish. Sci. 61: 684-686.
Cunningham, FX Jr, Chamovitz, D., Misawa, N., Gantt, E. and Hirschberg, J. 1993. Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta- carotene. FEBS Lett. 328: 130-138.
Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., and Miki, W. 1995. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene lebel.J. Bacteriol. 177: 6575-6584.
[0042]
[Example 2] Gene manipulation technology
Plasmid purification was performed using QIAprep (Qiagen). Ordinary genetic manipulation experiments such as restriction enzyme treatment, ligation reaction, transformation and the like were performed by the method shown in the above-mentioned Molecular Cloning of Sambrook et al. (1989).
[0043]
[Example 3] Preparation of chromosomal DNA from Flavobacterium genus A99-3
Flavobacterium (Flavobacteriumsp.) A99-3 strain (MBIC 03313) was cultured in 1,000 ml of Marine Broth (MB) medium (Difco) at 20 ° C. for 3 days. After collecting the cells, they were washed with STE buffer (100 mM NaCl, 10 mM TrisCl, 1 mM EDTA, pH 8.0), heat-treated at 68 ° C. for 15 minutes, 5 mg / ml lysozyme and 100 μg / It was suspended in I solution (50 mM glucose, 25 mM TrisCl, 10 mM EDTA, pH 8) containing ml of RNase A. After incubating at 37 ° C. for 1 hour, proteinase K was added to 250 μg / ml and incubated at 37 ° C. for 10 minutes. Further, N-Lauroylsarcosine · Na was added so that the final concentration was 1%, mixed well, and then incubated at 37 ° C. for 1 hour. Furthermore, after several phenol / chloroform extractions, the precipitated chromosomal DNA was wrapped around a glass rod while slowly adding twice the amount of ethanol, rinsed with 70% ethanol, and dissolved in 2 ml of TE buffer. Thus, a chromosomal DNA preparation was obtained.
[0044]
[Example 4] Preparation of cosmid library
The experimental procedure up to obtaining phage particles from the chromosomal DNA preparation was performed according to the instruction manual for the SuperCos 1 Cosmid Vector Kit manufactured by Stratagene. That is, the chromosomal DNA prepared from Flavobacterium sp.SauPartially decomposed with 3AI, cosmid vector SuperCos 1BamLigated to HI site and packaged into phage particles using LAMBDA INN (Wako, Osaka, Japan). And Erwinia UredoboracrtE,crtB,crtI E. coli carrying the lycopene-producing plasmid pACCRT-EIB containing the gene (E. coli) XL1-Blue MR strain was infected with the phage, and 1,000 colonies resistant to both Ap and Cm were obtained on LB plates containing Ap and Cm.
[0045]
[Example 5] Isolation of lycopene β-monocyclase gene
Escherichia coli XL1-Blue MR strain carrying pACCRT-EIB (including a gene capable of synthesizing lycopene from medium components), and DNA containing lycopene β-monocyclase gene of Flavobacterium genus A99-3 If the fragment was introduced and expressed by the SuperCos 1 vector, this clone should further metabolize lycopene to produce γ-carotene. Lycopene has a pink color, but γ-carotene has a yellow color (orange), so there are 1,000 yellow colonies that are resistant to both Ap and Cm on the plate. I chose 300. Since the color change was unclear, high-performance liquid chromatography (HPLC) with detection of a photodiode array (PDA) using a liquid culture of these E. coli cells and acetone-extracted cells. Analysis was performed (details are described in Example 8). From the HPLC retention time (33.1 minutes) and the UV-visible absorption spectrum (λmax, 434, 465, 494 nm) (Jeffrey et al, 1997), 3 clones considered to produce γ-carotene were obtained. Incidentally, the retention times of lycopene and β-carotene under these conditions were 23.6 minutes and 46.6 minutes, respectively.
[0046]
Flavobacterium genus (included in 1 of 3 clones)Flavobacterium sp.) A plasmid containing a 40 kb DNA fragment derived from the A99-1 strain was named pSC0601 and used for further analysis.
[0047]
Jeffrey, S. W., Mantoura, R. F. C., and Wright, S. W. (edited). 1997. Phytoplankton pigments in oceanography: Guidelines to Modern Methods.UNESCO Publishing.
[0048]
[Example 6] Subcloning and determination of nucleotide sequence
After digesting the inserted DNA fragment of plasmid pSC0601 with several restriction enzymes, pBluescript II KS^-(Toyobo) and transformed into E. coli JM109. It was confirmed by HPLC analysis of Example 8 that several clones were active, using these Escherichia coli liquid-cultured and cells extracted with acetone. One of them, 4.7 kbSalI-SacI fragment is pBluescript II KS^-ofSalI-SacThe plasmid inserted into the I site was named pBS0603, and the base sequence of this DNA fragment was determined. A DNA sequencing kit (Dye Terminator Cycle Sequence; Perkin-Elmer) and a model 3700 DNA sequencer (Perkin-Elmer) were used according to the attached protocol to determine the base sequence of the DNA fragment (4.7 kb) in both directions. The determined nucleotide sequence was analyzed using Sequencher, version 3.0 (Gene Codes Corporation), and the homology search of the confirmed ORF was performed by BLAST (Altschul and Lipman, 1990).
[0049]
As a result, the lycopene β-monocyclase gene (crtY- γ1134 bp open reading frame (ORF) was found encoding 377 amino acid sequences. The 377 amino acid sequence encoded by this gene is the existing Paracoccus (Paracoccus sp.) MBIC01143 strain (Agrobacterium aurantiacum) And Erwinia Uredobora (Erwinia uredovora) And other lycopene β-cyclase (CrtY: an enzyme that cyclizes both ends of lycopene) (Misawa et al, 1995). However, the degree of homology was low. That is, this Flavobacterium genus (Flavobacterium sp.) A99-1 lycopene β-monocyclase (CrtY-γ) is 22% and 19% identity with Paracoccus sp. MBIC01143 and Erwinia uredobora lycopene β-cyclase (CrtY), respectively. ).
[0050]
Furthermore, the above lycopene β-monocyclase (crtY- γ) Next to the gene, a 720 bp open reading frame (ORF) encoding a 239 amino acid sequence was found. The 239 amino acid sequence encoded by this gene is the existing Rhodobacter (Rhodobacter) It had homology over the entire amino acid sequence with CrtA [2'-monooxygenase] (Armstrong et al, 1989) such as genus bacteria. This was discovered this time as a gene that enhances lycopene β-monocyclase activity.
[0051]
Altschul, S. F. and Lipman, D. J. 1990. Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. USA 87: 5509-5513.
Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., and Miki, W. 1995. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.J. Bacteriol. 177: 6575-6584.
Armstrong, G. A., Alberti, M., Leach, F., and Hearst, J. E. 1989. Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster ofRhodobacter capsulatusMol. Gen. Genet. 216: 254-268.
[0052]
[Example 7] Preparation of plasmid for dye analysis
Using the plasmid pBS0603 obtained in Example 6 as a material, the following two types of plasmids were prepared for dye analysis.
pBS0603 to 1.54 kbEcoRV-EcoExcept for the RI fragment, instead,EcoRI-NotI-BamHI adapter (BamA plasmid in which HI terminal side was smooth) (Takara) was prepared and named pBS0603dDO. This plasmid contains pBluescript II KS^-The vector contains Flavobacterium A99-3crtY- γWhencrtA2 genes are inserted. Next, lycopene β-monocyclase (crtY- γ) A plasmid (pG0604) for expressing only the gene in E. coli was prepared as follows. Oligonucleotide 5 'TGTCTGCAGGATTTCAACCGTATTTGATT 3' and 5 'TATGGATCCCAATTCTTCAATCTTAAAGGAG 3' using plasmid pSC0601 as templatecrtY- γThe gene was amplified by PCR, and the obtained amplification product was cloned into a pGEM-T Easy vector (manufactured by Promega) according to the instruction manual for this vector. PCR was performed at 94 ° C. for 10 minutes, followed by 40 repetitions of 94 ° C. for 1 minute, 50 ° C. for 1 minute, 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 10 minutes.
[0053]
[Example 8] Extraction and analysis of carotenoid accumulated in E. coli
E. coli containing plasmid pACCRT-EIB (E. coli) Accumulates lycopene in the cell. E. coli containing cosmids from pACCRT-EIB and SuperCos 1 (E. coli) XL1-Blue MR or pACCRT-EIB and pBluescript II KS^-E. coli JM109 containing the plasmid subcloned by E. coli, or E. coli JM109 containing pACCRT-EIB and pG0604 were cultured with shaking in LB medium containing Cm and Ap for 2 days at 30 ° C. (100 rpm). Then, the cells were collected from the culture solution, added with acetone, and stirred at room temperature for 30 minutes. The extract was centrifuged at 14,000 x g for 5 minutes (4 ° C), and the supernatant, which was a carotenoid pigment extract, was subjected to HPLC (model 2695, manufactured by Waters) analysis. Conditions are 25 ° C and reverse phase column (3.9 x 300 mm, Nova-pak HR 6 μm C₁₈; Waters model 2996 PDA detection using acetonitrile (acetonitrile) -methanol (2-propanol) (90: 6: 4) and developing at a rate of 1 ml / min. Monitored by instrument.
[0054]
Plasmid pBS0603dDO (crtY- γWhencrtA) Or pG0604 (crtY- γE. coli was introduced together with pACCRT-EIB, and liquid culture was performed, and HPLC analysis was performed using the cells obtained by acetone extraction. As a result, the total amount of carotenoids of both was almost the same, but the former synthesized 85% γ-carotene and 15% β-carotene, while the latter synthesized 40% γ-carotene and 60%. % Β-carotene was synthesized (see Example 9 for identification of γ-carotene). In both cases, almost no substrate lycopene remained. From the above results, it was found that CrtY-γ produces γ-carotene from lycopene, but depending on the culture conditions, part of the generated γ-carotene can be converted to β-carotene. In addition, when CrtA coexists with CrtY-γ, the amount of γ-carotene synthesized increased, indicating that CrtA of Flavobacterium A99-3 strain enhances lycopene β-monocyclase activity. Note that the abovecrtY- γInstead of genes, the Erwinia bacteriacrtYWhen the gene is used together with pACCRT-EIB, almost 100% β-carotene is obtained and γ-carotene cannot be obtained under the same conditions as above.
[0055]
[Example 9] Identification of γ-carotene
Plasmid pBS0603dDO was transformed into E. coli containing pACCRT-EIB (E. coli) Introduced into the JM109 strain and cultured in 2,000 ml of LB medium containing Ap and Cm at 30 ° C. for 2 days (100 rpm). Cells collected from the culture were extracted with 300 ml of acetone. This was concentrated and extracted twice with 200 ml of hexane. Further, the hexane layer was concentrated, subjected to silica gel column chromatography, and developed and eluted with hexane-ethyl acetate (50: 1) to obtain 1 mg of a pure product considered to be γ-carotene.
[0056]
UV-visible absorption spectrum, FD-MS spectrum (Hitachi double-focusing mass spectrometer M-2500) (m / e 536), and¹This product was identified as γ-carotene (β, ψ-carotene) by 1 H-NMR (500 MHz, JEOLα) spectrum.
[0057]
【The invention's effect】
The present invention makes it possible to produce a large amount of monocyclic carotenoids that exist only in a small amount in nature.
[0058]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing functions and biosynthetic pathways of various carotenoid synthesis genes in Erwinia bacteria and Paracoccus bacteria.
[Fig. 2] Marine bacterium Flavobacterium (Flavobacteriumsp.) Diagram showing the functions of various carotenoid synthesis genes in A99-3 strain and the presumed carotenoid biosynthetic pathway.

Claims (8)

A gene encoding the peptide shown in the following (a), (b), or (c):
(A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B) a peptide having an lycopene β-monocyclase activity, comprising an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2;
(C) a peptide derived from a bacterium encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA that hybridizes with the DNA complementary thereto under stringent conditions, and having a lycopene β-monocyclase activity .   A microorganism obtained by introducing the gene according to claim 1 together with another carotenoid biosynthesis gene.   A microorganism obtained by introducing the gene according to claim 1 into a microorganism containing another carotenoid biosynthesis gene. The microorganism obtained by further introducing the gene encoding the peptide shown in the following (d), (e), or (f) into the microorganism according to claim 2 or 3:
(D) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4,
(E) a peptide consisting of an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 4 and improving the yield of γ-carotene ;
(F) a peptide derived from a bacterium encoded by DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 or DNA complementary thereto and hybridized under stringent conditions, and improving the yield of γ-carotene .   A method for producing a monocyclic carotenoid, wherein the microorganism according to any one of claims 2 to 4 is cultured in a medium to obtain a monocyclic carotenoid from the culture or the microbial cell.   The method for producing a monocyclic carotenoid according to claim 5, wherein the microorganism is Escherichia coli.   The method for producing a monocyclic carotenoid according to claim 5, wherein the other carotenoid biosynthetic genes include at least a gene group required for synthesizing lycopene from farnesyl pyrophosphate or geranylgeranyl pyrophosphate. Peptides shown in the following (a), (b), or (c):
(A) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B) a peptide having an lycopene β-monocyclase activity, comprising an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2;
(C) a peptide derived from a bacterium encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA that hybridizes with the DNA complementary thereto under stringent conditions, and having a lycopene β-monocyclase activity .

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