TW200407430A - Cells secreting R-α- lipoic acid and method for fermentative production of said R-α- lipoic acid - Google Patents
Cells secreting R-α- lipoic acid and method for fermentative production of said R-α- lipoic acid Download PDFInfo
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Description
200407430 五、發明說明（1) 一、【發明所屬之技術領域】 在許多原核生物及真核生物中R - 〇：-硫辛酸係特殊多 酶複合物之重要輔因子。R- α -硫辛酸總是共價鍵結在適 當酶之特定離胺酸殘基之ε -胺基上。如此，R - α -硫辛酸 係丙酮酸脫氫酶（P DH) [ E C 2 · 3 · 1 · 1 2 ]及α -酮戊二酸脫氫 酶（KGDH)[EC 2· 3· 1· 61 ] Ε2次單元之一部分，並在該處扮 演一個重要角色，在α -酮酸氧化脫羧作用中作為氧化還 原伙伴及醯基供體。再者，在胺基乙酸解離酶系統内硫辛 酸係胺基甲基之載體。 α -硫辛酸係一光活性分子，其對掌性中心位於c 6碳 原子上。α -硫辛酸之R構形係天生之鏡像對映體。唯有此 種形態具有生理活性作為對應酶之輔因子。—硫辛酸可 呈氧化悲（5 - [ 1，2 ]-二氫二噻ρ戊—3 -基戊酸）及還原態（β，8〜 二氫硫基辛酸）存在。’’ α ~硫辛酸”一詞以下係指· α —硫辛 酸之該兩種形態及其特殊鹽類，例如··鈣、鉀、鎂、納 銨之鹽。 / R- α -硫辛酸之生物合成業經特別密集地對大腸桿 細菌加以研究（請參閱第一圖）。此處，在硫辛酸合 與酿載體蛋白質UCP)共價鍵結之辛酸係一特定前體。 一複雜反應中，將兩個硫原子轉移到經活化之辛酸（辛辟 acp)上，ϊ「ΐΓα_硫辛酸—acp。該反應係由硫轉移i二 辛酸合f酶[EC 2.8.卜]llpA基因產物催化。胺基酸‘ 胱虱酸取伋作為硫供體。隨後R…硫辛酸由r_200407430 V. Description of the invention (1) 1. [Technical field to which the invention belongs] In many prokaryotes and eukaryotes, R-0: -lipoic acid is an important cofactor for special multi-enzyme complexes. R-α-lipoic acid is always covalently bonded to the ε-amine group of a specific lysine residue of an appropriate enzyme. Thus, R-α-lipoic acid pyruvate dehydrogenase (P DH) [EC 2 · 3 · 1 · 1 2] and α-ketoglutarate dehydrogenase (KGDH) [EC 2 · 3 · 1 · 61] Part of the E2 subunit, and plays an important role there, as a redox partner and a fluorenyl donor in the oxidative decarboxylation of α-keto acids. Furthermore, a lipoic acid-based aminomethyl carrier is used in the aminoacetic acid dissociation enzyme system. Alpha-lipoic acid is a photoactive molecule with its palmar center on the C 6 carbon atom. The R configuration of α-lipoic acid is a natural mirror image enantiomer. Only this form has physiological activity as a cofactor for the corresponding enzyme. —Lipoic acid can exist in oxidized state (5- [1,2] -dihydrodithia-p-penta-3-ylpentanoic acid) and reduced state (β, 8 ~ dihydrothiocaprylic acid). The term "α ~ lipoic acid" refers to the two forms of α-lipoic acid and their special salts, such as the salts of calcium, potassium, magnesium, and sodium ammonium. / R-α-lipoic acid The biosynthesis industry has particularly intensively studied coliform bacteria (see the first figure). Here, caprylic acid, a covalent bond of lipoic acid and the carrier protein UCP, is a specific precursor. In a complex reaction, Two sulfur atoms were transferred to an activated octanoic acid (octaprate), and "ϊαα-lipoic acid-acp. This reaction was catalyzed by the thiotransfer i-caprylic acid fase [EC 2.8. Bu] llpA gene product. The amino acid 'cystic acid is extracted as a sulfur donor. Then R ... lipoic acid is obtained from r_
-ACP轉移至α-酮酸脫氫酶之E2次單元係藉硫辛酸蛋白辛質I-ACP transfer to the α-keto acid dehydrogenase E2 subunit is lipoic acid protein octane I
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200407430 五、發明說明（2) 連接酶B [EC 6. 催化，因不含R〜a〜护 R- a-硫辛酸，lipB基因產物呈自由中間ς醢-ACP或 2000，生物化學39 :15166至15178)。 米勒等人， 有關R - a -硫辛酸在真核生物中之生人 。但’ R: a -硫辛酸合成及轉移至對應酶係：照：知『多+ 内之方式在真核生物細胞粒線體内實施。“ ' 以、、、田囷 除其係新陳代謝中擔任主要角色之酶之 不久以前亦對^硫辛酸對藥物治 要成为外， NUtraCeutlcal)之重要性加以肯定，由於為二物補品（200407430 V. Description of the invention (2) Ligase B [EC 6. Catalysis, because it does not contain R ~ a ~ R-a-lipoic acid, the lipB gene product is free intermediate 醢 A-ACP or 2000, Biochemistry 39: 15166 To 15178). Miller et al., Regarding the birth of R-a-lipoic acid in eukaryotes. But ‘R: a -lipoic acid is synthesized and transferred to the corresponding enzyme system: Photo: The method of“ ++ inside ”is implemented in the eukaryotic cell mitochondria. "'In addition to the enzymes that play a major role in metabolism, not long ago, the importance of ^ lipoic acid in the treatment of drugs, NUtraCeutlcal) was affirmed.
, 0，bV；A 受氧化應力所引發之有害加工二rn:3 酸（α—硫辛酸之還原態）在體内⑴可直接或接m辛 =化之天然抗氧化劑，例如：抗：門=生其他 生育酚（維生素E)或若缺乏該等維生素時If素Y)或;1 抗氧化劑（由於豆作A強、f' 可曰代4 4 八作為強遮原劑之特性）。所以，在與抗壞 血酸、α-生育酚及穀胱甘肽（"抗氧化劑網"）作用時、，j 一 奴辛酸特別重要。α —硫辛酸亦可用以預防及控制第Η型 糖尿病及破壞其副作用（例如：多神經病、白内障或心臟 血管狀況）。 雖然有更多跡象顯示：與3形態者相較，純R形態α 一 硫辛酸之用途具有顯著優點，目前，該兩種α —硫辛酸鏡 像對映體之不同生物學活性仍是密集研究之目標。因此， ^ 體外（5式管内）實驗顯示，僅天然R - α -硫辛酸可形成 Β能性α -酮酸脫氩酶。相反地，S鏡像對映體對R— α -硫, 0, bV; A Harmful processing of rn: 3 acid (reduced state of α-lipoic acid) caused by oxidative stress In the body, it can be directly or directly connected to natural antioxidants such as: = To produce other tocopherols (vitamin E) or If element Y in the absence of these vitamins); 1 antioxidant (because beans are strong A, f 'can be said to be 4 4 8 as a strong masking agent). Therefore, when used with ascorbic acid, α-tocopherol and glutathione (" Antioxidant Network "), j-noctanoic acid is particularly important. Alpha-lipoic acid can also be used to prevent and control type diabetes and destroy its side effects (eg, polyneuropathy, cataracts or cardiovascular conditions). Although there are more indications that compared with the three forms, the use of pure R form alpha monolipoic acid has significant advantages. At present, the different biological activities of the two α-lipoic acid mirror image enantiomers are still intensive research. aims. Therefore, in vitro (in a tube of type 5) experiments have shown that only natural R-α-lipoic acid can form beta-functional α-keto acid deargonase. In contrast, the S mirror image enantiomer pair R-α-sulfur
200407430 五、發明說明（3) Ϊ : : ί f it S::性同化作用甚至具有抑制作用。α -硫 之再生ί = 粒線體内抗氧化活性α —二氣硫辛酸 為重要。與r鏡像對映體結合時哺乳動 辛龄胺ϊϊί私草醯胺腺。票呤二核苔酸（ναμ)相關硫 ^ 活性較與s形態結合時者高出2〇倍。此外 兄像對映體相較，R_ α—硫辛酸對於抗胰島素老 二j島素景多響之葡萄！ t吸收Α骨骼肌細⑯葡萄糖新陳代 f二有顯著更強烈作用。再者，在實施動物實驗者，R形 悲者具有消炎作用，而S形態者則具有止痛作用。所以， 為避免不必要之副作用，總是極希望能提供僅具有鏡像對 映體純形態之α —硫辛酸。 一、【先前技術】 目前，α -硫辛酸之工業製造唯獨採用化學方法，最 終產品均係R形態及S形態之消旋物（亞達夫等人，1 99〇， 科學工業研究學報49 :400至409)。為製得鏡像對映體純 R - α -硫辛酸，曾研發出許多種方法。舉例言之，藉助於 對掌性輔助劑之化學作用（華爾頓等人，1 9 54，美國化學 學會學報76 : 4748 ;德國專利DE 4 1 37773 )或藉酶的作用 (愛德格等人，1 9 9 5，化學學會化學報導學報·· 1 5 6 3至 1 5 6 4 )可辨別α —硫辛酸或合成中間產物中一個之消旋物。 換言之，為防止由於一個鏡像對映體合成步驟而形成消旋 物，可利用化學作用（DE 3 6 2 9 1 1 6 ; DE 1 9 5 3 388 1 ;布瑞英 曼等人，1 9 9 9，自然研究學報54b : 6 5 5至661 ; DE 1 0 0 3 6 5 1 6 )或藉助於微生物利用立體特異生物轉化作用（哥 ZUU^fU/^^υ 五、發明說明（4) 帕蘭及雅格布斯，1 g 8 沙拉第等人，199〇 , ^，四面體通訊30 :5705至5708 ;達 DE 1 0 0 5 6 0 25 )可引、隹，學學會化學報導學報：729至73 0 ; 。依次，利用天生☆掌性中心 D -甘露糖醇，若干其手性反應物，例如：s '順丁烯二酸或 酸之化學合成（布魯古他方法已開始鏡像對映體純α —硫辛 • 兄，、斤及哥爾丁，1988，化學學會學報200407430 V. Description of the invention (3) Ϊ:: ί f it S :: Sexual assimilation even inhibits. Regeneration of α-sulfur ί = Antioxidant activity in the mitochondria α-digasic lipoic acid is important. Breasting when combined with the enantiomer of the r mirror image. The activity of sulfuric acid related to oxaline dinuclear acid (ναμ) is 20 times higher than that when combined with s-form. In addition, compared to the enantiomers of the brothers, R_α-lipoic acid is a grape that is louder than the insulin resistant second j island prime! t absorption of skeletal muscle cells, glucose, and new generation f2 have significantly more potent effects. Furthermore, in animal experimenters, the R-shaped sorrowful person has anti-inflammatory effect, while the S-shaped person has analgesic effect. Therefore, in order to avoid unnecessary side effects, it is always highly desirable to provide α-lipoic acid in a purely enantiomeric form. I. [Previous Technology] At present, the industrial production of α-lipoic acid uses only chemical methods, and the final products are all racemates in the R form and S form (Yadav et al., 1 99, Journal of Scientific and Industrial Research 49: 400 to 409). A number of methods have been developed to prepare the enantiomerically pure R-α-lipoic acid. By way of example, by means of a chemical action on palmitious adjuvants (Walton et al., 195, Journal of the American Chemical Society 76: 4748; German patent DE 4 1 37773) or by the action of an enzyme (Edg et al. Human, 1 995, Journal of Chemical Reports of the Chemical Society ... 1 5 6 3 to 1 5 6 4) Distinguish α-lipoic acid or one of the synthetic intermediates. In other words, in order to prevent the formation of racemates due to a mirror image enantiomer synthesis step, chemical action can be used (DE 3 6 2 9 1 1 6; DE 1 9 5 3 388 1; Bryingmann et al., 1 9 9 9. Journal of Natural Research 54b: 6 5 5 to 661; DE 1 0 0 3 6 5 1 6) or the use of stereospecific biotransformation with the help of microorganisms (Brother ZUU ^ fU / ^^ υ V. Description of the invention (4) Pa Lan and Jacobs, 1 g 8 Sarah et al., 199, ^, Tetrahedron Bulletin 30: 5705 to 5708; DE 1 0 0 5 6 0 25) Citation, 隹, Journal of Chemical Journal of Chinese Academy of Sciences: 729 to 73 0;. In turn, using the natural ☆ palm center D-mannitol, a number of its chiral reactants, such as: chemical synthesis of s' maleic acid or acid (the Bruguta method has begun to mirror the enantiomerically pure α — Lipo, Brother, Jin, and Golding, 1988, Journal of the Chemical Society
Perkin Trans· 1:9至12;拉瑪勞等人，1987，四面體通 说28 ’2183至2186)。由於部分合成步驟繁複、產率低且Perkin Trans. 1: 9-12; Ramalau et al., 1987, Tetrahedron Talk 28'2183-2186). Due to the complexity of some synthetic steps, low yield and
原料成本高，所有業經公開之製造鏡像對映體純r — α 一硫 辛酸之方法目前均不經濟。 表近’許多低分子量天然物質（例如：抗生素、維生 素或胺基酸）經常藉助於發酵方法利用微生物之不同菌株 實施工業生產。 利用可過表現一硫辛酸-合成酶基因（丨i ρΑ基因）之細 胞可達成此目的。 本發明之過表現最好意謂：與某一特定野生型細胞（ 自该野生型細胞業將該疏辛酸-合成酶基因分離出來）相較 ’硫辛酸-合成酶基因之表現至少高出2倍，尤以至少高出 5倍則更佳。The cost of raw materials is high, and all published methods for producing mirror-enantiomer pure r-α-lipoic acid are currently uneconomical. Apparently, many low-molecular-weight natural substances (such as antibiotics, vitamins, or amino acids) are often industrially produced by fermentation methods using different strains of microorganisms. This can be achieved using cells that can overexpress the monolipoic acid-synthetase gene (丨 i ρΑ gene). The best performance of the present invention means that the performance of the lipoic acid-synthetase gene is at least 2 higher than that of a specific wild-type cell from which the lipoic acid-synthetase gene is isolated. Times, especially at least 5 times higher.
該硫辛酸-合成酶基因最好係一具有順序識別號碼：1 或該基因之官能變體。 本發明之官能變體係指：一 D Ν Α順序，該D Ν Α順序係藉 核苷酸之消除、插入或取代自順序識別號碼：1中所圖示 之順序衍生出來，而硫辛酸-合成酶之酶活性係由保留之The lipoic acid-synthase gene is preferably a functional variant having a sequential identification number: 1 or the gene. The functional change system of the present invention refers to: a D Ν Α sequence, which is derived from the sequence shown in the sequence identification number: 1 by the elimination, insertion or substitution of nucleotides, and lipoic acid-synthesis Enzymatic activity
第7頁 200407430 五、發明說明（5) 基因編碼。 為過表現該細胞内之1 i PA基因，一 有-增加之lipA基因複製數目及/或加T在表現中可具 其由於適當啟動子。 X日加之ΗΡΑ基因，尤 1 1 PA基因之過表現總是以至少 硫辛酸-合成酶活性。 ，、L數增加細胞之 隶好’本發明之/個細胞過表一 基因，該基因編碼—蛋白質，該蛋白質勺=酸：合成酶 :2或順序超過4Q% #於順序 巧。項序識別號碼 同源於順序識別號瑪：2之順序^超過^官 = 土變體/ 超過8 0 %更佳。 b (U為佳，尤以 在本發明中，所述所有同源值係指利用最 去（GCG威斯康辛套裝軟體，遺傳電腦組 斯康辛）所得之結果。 ）麥迪遜，威 細胞内一 1 ipA基因之複製數目可利用技術精 曾加。所以，舉例言之，可將_1ιρΑ&因貝】 有母個細胞多重複製品（例如：在大腸桿菌之突 =t : ^c19、PBR322、pACYC184)t質體栽體並將該基木因 弓丨進細胞内。另—變通方式是，一 lipA基因之多 。 可整合在細胞之染色體内。可用之整合方法係習知 ，该等系統係利用溫和噬菌體、整合性質體调* 組之整合(例如：漢彌頓等人，細菌學報，71 :、二同至原重 4 b 2 2 ) 〇 、在啟動子控制下藉選殖（無性繁殖）一 1 i p A基因至一Page 7 200407430 V. Description of the invention (5) Gene coding. In order to overexpress the 1 PA gene in the cell, there is an increase in the number of copies of the lipA gene and / or the addition of T in the expression may be due to a suitable promoter. X-days, together with the overexpression of the HPA gene, especially the 1 1 PA gene, always show at least lipoic acid-synthase activity. The number of cells with an increased L number is better than the one of the cells of the present invention. The gene encodes a protein, the protein scoop = acid: synthetase: 2 or more than 4Q%. The item sequence identification number is the same as the sequence identification number Ma: the order of 2 ^ over ^ guan = soil variant / more than 80% is better. b (U is better, especially in the present invention, all homology values refer to the results obtained by using the most (GCG Wisconsin software package, genetic computer group Sconsin).) Madison, Wei cell intracellular-1 The number of copies of the ipA gene can be increased using techniques. So, for example, _1ιρΑ & Imbe] can have multiple replicates of mother cells (for example: in E. coli = t: ^ c19, PBR322, pACYC184) t plastid plant and use the basic wood factor Arch into the cell. Another-workaround is as many lipA genes as possible. Can be integrated into the chromosome of the cell. Available integration methods are known. These systems use the integration of mild bacteriophages, integrated integrations * (eg, Hamilton, et al., Acta Bacterium, 71 :, Ertong to original weight 4 b 2 2). Borrowing (asexual reproduction)-1 ip A gene to-under the control of the promoter
200407430 五、發明說明（6) 一 質體載體内而增加複製數目為佳。尤以藉 至一 PUC衍生物内[例如：pASK—IBA3 ( IBA，生八=土介因 所:哥廷根，德國）]而增加大腸桿菌内之複製數刀目 九。 所以本發明之另一内容係一曾，一 衣 更{土。 下該質體含有一lipA基因。 ' — 動子之官能控制 基因之天然啟動子及操縱基因 碼1 1ΡΑ基因表現之控制區亦屬 =貝體-編 啟動子upA基因之表現亦可增加。可# 助於其他 硫辛酸-合成酶基因（例如：g A美组，、$式或控制式 氫酶啟動子或可誘導lac、tae、、、、./刀甘油醛磷酸去 大腸桿菌啟動子）可誘導表現之適杳啟:^ : Ka或tet 項技術者所習知者（馬克 " 系統係精於此 512^ 5 38 ) 〇 訢，1 9 96，微生物評論6〇 : 。 以結構可以習知方式用在質體或染色體上 在一特別合適之具體實施例 需啟動子之質體（例+ ^ 3有提升表現所 礙物系統）係用以J殖园之可誘導tet啟動子/阻 位或基：的= 如··核糖核蛋白體結合部 出現或藉用比較常發生的贫u建構上的最適化順序）之 較鞴Φ的玄 吊^生的始碼子代替依昭Π宓雜7 + 車乂稀^的松螞子均可提升表現。9 ^…在螞子使用"比 本發日月Μ 嗜等規# w：、細胞最好含有—個包括一 1 i pA A 。亥4規律性訊號之該等修飾。 UpA基因之質體及 舉例言之 “ 200407430 五、發明說明（7) —— 1 ipA基因及隨後與載體DNA碎片連接之特^ 可將一lipA基因選殖入一質體載體内。、疋引物（先導物） 本發明之若干細胞（其HpA基因之表現 者增加且同時增加硫辛酸—合成酶活性 起始細皰 生物技術由一起始細胞製得。 用^準分子〜 硫辛酸—合成酶基因f、經在許多細胞 电明之細胞最好由原核生物或真核生物 1疋。因此本 可t成Ri-硫辛酸本身（起始細 1胞，該等細跑 組法且可藉發酵作用培養。 ，/專細胞可採用重 之植物或動物細胞亦 在細胞培養物 本發明之細胞以二:本發明之細胞。 長 仁以％内桿菌 甘A、、、田囷囷株） 株最=。 园囷株較佳’尤以大腸桿菌種：； 體引進一起始細巧阻力，將含有lipA ，方法（例如：電穿透作^儲臧貝體之種系係使用—普通轉 二、【發明内容】） 本發明之内容係八、 細胞以發酵方式制^ ▲刀泌r— α—硫辛酸之細胞及利用該等 本發明之内==該[α—硫辛酸之方法。 映體純R- α ~碗辛^ ’八提供若干細胞，該等細胞可將鏡像對 本發明之另―久刀泌在培養基内。 包括本發明之質各疋製備本發明細胞之方法，該方法 ^ 7| z; , 本發明之另 芝一起始細胞。 的係提供一種發酵方法，該方法可製 第10頁 200407430200407430 V. Description of the invention (6)-It is better to increase the number of replication in the plastid vector. In particular, borrowing a PUC derivative [for example: pASK-IBA3 (IBA, raw eight = Tosukeinori: Göttingen, Germany)] to increase the number of copies in E. coli. Therefore, another aspect of the present invention is once. The plastid contains a lipA gene. '— Functional control of the mover The natural promoter and operator of the gene code 1 The control region for the expression of the 1PA gene also belongs to = corpuscle-edit The expression of the promoter upA gene can also be increased. May # help other lipoic acid-synthetase genes (for example: g A US group, $ or controlled hydrogenase promoter or inducible lac, tae, ,,, /. Knife glyceraldehyde phosphate de-E. Coli promoter ) Revelation of inducible performance: ^: Known by Ka or tet technicians (Mark " system is good at this 512 ^ 5 38) 〇xin, 196, Microbiological Review 60 :. The structure can be used in a conventional manner on plastids or chromosomes. In a particularly suitable embodiment, the plastids that require a promoter (eg + ^ 3 has an obstacle to improve performance system) are used to induce the tet of J colony. Promoters / resistances or groups: = such as · ribonucleosome binding site appears or borrows the most optimized sequence of poor construction (the optimal sequence of construction)) instead of the original code According to Zhao 宓 宓 7+ 乂 乂 ^^ 松松 子 can improve performance. 9 ^ ... Used in ants. "The present day and month M homophile #w: The cell should preferably contain-1 i pA A. These modifications of the Hai 4 regular signal. The plastid of the UpA gene and the example "200407430 V. Description of the invention (7) —— 1 ipA gene and the characteristics of subsequent linking with the vector DNA fragment ^ A lipA gene can be selected into a plastid vector. (Leader) Several cells of the present invention (the expression of HpA gene increases and at the same time increases lipoic acid-synthetase activity. Initiating fine vesicle biotechnology is made from a starting cell. Using excimer ~ lipoic acid-synthetase gene f. The cells that have been electrolyzed in many cells are preferably made of prokaryotes or eukaryotes. Therefore, they can be converted into Ri-lipoic acid itself (starting with 1 cell, these fine-run groups can be cultured by fermentation) The special cells can be heavy plant or animal cells also in the cell culture. The cells of the present invention are as follows: the cells of the present invention. Garden strains are better 'E. coli species: Introduce a starting fine resistance, will contain lipA, methods (for example: electro-penetration for the storage of germplasm of Zangbei shells-general transfer II, [contents of the invention 】) The content of the present invention is eight Cells are made by fermentation ^ ▲ blade r-α-lipoic acid cells and methods of using the present invention == the [α-lipoic acid. Enantiomerically pure R-α ~ bowl xin ^ 'eight provide several Cells, which can mirror the other of the present invention-long knife secreted in the culture medium. Including the method of the present invention to prepare the cells of the present invention, the method ^ 7 | z; The system provides a fermentation method, which can be made on page 10 200407430
造鏡像對映體純R- α -硫辛酸。 種包括將本發明 _ 化瑨養在一培養基Φ夕+、丄 Ά該目的'，該細胞分泌自由形態之鏡像對映體純R a k辛馱至培養基内，再將該鏡像對映护辛 自該培養基中取出。 α &辛酸 可用精於此項技術者習知 之離心作用取出細胞及藉產品 基中回收R- α -硫辛酸。 之方法（例如：實施培養基 之隨後萃取或沉澱）自培養 四、【實施方式】 本發明之方法係R- α -硫辛酸整個合成作用及利用活 細胞唯獨在一發酵程序内實施之第一種方法。盥傳統化興 方法比較，本發明方法之優點硫辛酸之鏡效對映干 體之特別生物合成，因此可省去合成鏈過程中或终止時繁 複之消旋體辨認程序。再者，本發明之卜α_硫辛酸f造、 方法對環境之傷害遠低於化學合成法。 生理及生化數據顯示硫辛酸存在於野生型細胞内實際 上經常呈結合形態，蓋因R-α-硫辛酸業已以完全蛋白質— 結合方式合成（赫伯特及蓋斯特’ 1 9 75，原始微生物學1〇6 :259至266 ·’米勒等人’2000 ’生物化學39 至 15178)。 ’ 但，驚奇的是，經發現在本發明之架構内一硫辛酸一 合成酶基因之過表現導致自由鏡像對映體純[^ _硫辛酸 積存在宿主生物之培養基内。如此’進而在生物質量業經 移除之後，可將生成物自培養基簡單地分離出來，'無需事Enantiomeric enantiomerically pure R-α-lipoic acid. The invention includes cultivating the present invention in a culture medium Φ Xi +, 丄 Ά the purpose ', the cell secretes a free-form mirror-image enantiomer pure Rak Xin into the culture medium, and then the mirror-image enantiomer Hu Xin from Remove the medium. α & Caprylic acid Cells can be removed by centrifugation known to those skilled in the art and R-α-lipoic acid can be recovered from the product base. Methods (for example: performing subsequent extraction or precipitation of the culture medium) for self-cultivation IV. [Embodiment] The method of the present invention is the first synthesis of R-α-lipoic acid and the use of living cells is the only one implemented in a fermentation process Methods. Compared with traditional methods, the advantages of the method of the present invention are the special biosynthesis of the enantiomer of lipoic acid, so that the complicated racemic identification procedure during or at the end of the synthesis chain can be omitted. Furthermore, the α-lipoic acid method and method of the present invention is far less harmful to the environment than the chemical synthesis method. Physiological and biochemical data show that lipoic acid is often present in wild-type cells in a bound form. Gein R-α-lipoic acid has been synthesized in a complete protein-binding manner (Herbert and Geist '1 9 75, original Microbiology 106: 259 to 266. 'Miller et al. 2000' Biochemistry 39 to 15178). However, it is surprising that the overexpression of a lipoic acid-synthetase gene in the framework of the present invention has resulted in the free mirror-image enantiomerically pure [^ _lipoic acid accumulating in the host organism's culture medium. In this way, and after the biological mass has been removed, the product can be simply separated from the culture medium,
200407430 五、發明說明（9) 先破壞細胞或藉助於一繁複上 載體蛋白質將該R- « -硫辛萨兩成本水解步驟自其結合之 E2次單元）。 ·夕除（ACP或α -酮酸脫氫酶之 本發明用以製造α〜硫 公開之微量鹽培養基内（赫伯牡咬=、，、田胞最好培養在文獻 學18Δ、269至272)。 、、及盍斯特，1970，理論酶 原則上，可使用任何可 類作為碳源。以使用天夂气萨=糖類、糖醇類或有機酸 、反丁烯二酸、麩酸、葡；：：頻果酸、琥錢、丙嗣酸 尤以號ίέ酸及酸草醯乙酸更卩$油或酸轉乙酸為佳。 組合體亦屬可能。再者，；：表=由數個碳源所形成之 / C； ΑΦ Z； PQ ^ ^ ^養基内添加具有鏈長C2至C8 、、 更佳）之短鏈脂肪酸（分別為己酸及辛 酸）作為α-硫辛酸合成之特定前驅物則亦屬可能。 源之濃度以1至30公克/公升為佳。 Α 本發明之細胞最好在右@ +立莫卩主、口 t / 、 ％ τ你令虱培養情況下及特定細胞最適 生長溫度範圍内保溫1 6至1 5 〇小時。 最適溫度範圍以1 5至5 5 °C為佳，尤以3 0至3 7 °C更佳。 舉例言之，藉助於生物學鑑定利用一硫辛酸營養缺陷 蜇指示菌株（lipA突變體）可將本發明方法所製r— 硫辛 酸加以偵檢及量化。此種R— α —硫辛酸之比濁量化作用業 經文獻公開（赫伯特及蓋斯特，1 9 7 〇，理論酶學丨8 A，2 6 9 至2 7 2 )。但，除葡萄糖之外，若培養基亦含有乙酸脂及號 珀酸酯，本發明架構内所用指示蘭株” 485 1 ip2 (ATCC 2 5 6 4 5 )亦將生長，無需補充之r — α —硫辛酸。當測定所製200407430 V. Description of the invention (9) The cell is destroyed first or the complex of the carrier protein is used to combine the R-«-lipinza into two hydrolysis units (E2 subunit). · Epidemium (ACP or α-keto acid dehydrogenase of the present invention is used to produce α ~ sulfur disclosed micro-salt medium (Herber's Bite = ,,, and field cells are best cultivated in literature 18Δ, 269 to 272 ). ,, and Zest, 1970, theoretical enzymes, in principle, any kind can be used as a carbon source. In order to use natural gas gas = sugars, sugar alcohols or organic acids, fumaric acid, glutamic acid, glucose ; :: Fructoic acid, succinic acid, and propionic acid are more preferred, and acetic acid and acetic acid are more suitable for oil or acid-to-acetic acid. Combinations are also possible. Moreover,;: == by several Carbon source formed / C; ΑΦ Z; PQ ^ ^ ^ added short-chain fatty acids (respectively hexanoic acid and caprylic acid) with chain lengths C2 to C8, and better in the nutrient as specific for the synthesis of α-lipoic acid Precursors are also possible. The concentration of the source is preferably 1 to 30 g / liter. Α The cells of the present invention are preferably incubated for 16 to 150 hours at the right @ + 立 莫 卩 主, 口 t /,% τ in the case of lice culture and the optimum growth temperature range of the specific cell. The optimum temperature range is preferably 15 to 55 ° C, and more preferably 30 to 37 ° C. For example, the use of a lipoic acid auxotroph 蜇 indicator strain (lipA mutant) by means of biological identification can detect and quantify the r-lipoic acid produced by the method of the present invention. The turbidity quantification of such R-α-lipoic acid has been published in the literature (Herbert and Geist, 1970, Theoretical Enzymology, 8A, 26.9 to 27.2). However, in addition to glucose, if the culture medium also contains acetate and succinate, the indicator orchid strain "485 1 ip2 (ATCC 2 5 6 4 5)" used in the framework of the present invention will also grow without the need to supplement r — α — Lipoic acid
第12頁 200407430 五、發明說明（10) R a ~辛酸時’為防止生物學鑑定中該指示菌株之假〜正 生長亥生長係由於（例如）：除R — α -硫辛酸之外，並引進 由產體（生產者）菌株分泌之葡萄糖及乙酸與琥珀酸，即使 R - -硫/辛酸產體之生長最好用琥珀酸酯作為唯一碳源。 違菌，係補充以本發明細胞培養物之上清液；隨後可根據 指示菌株生長以測定該培養基内之硫辛酸含量。 ，—炫利用下列諸實驗例將本發明作進一步說明。實施該 等貫驗例所用之細菌菌株大腸桿菌”丨^/口八“一丨“^丨丨^ 曾依照布達佩斯條約以編號DSM 1 5 1 04寄存在德國微生物 及培養細胞收集公司、勃朗希威格，德國）。 貫驗例1 : pASK - I ΒΑ3 - 1 i ρΑ載體（媒介生物）之建造 Α· 1 ipA基因放大 藉助於聚合酶鏈反應（p C R)利用p w 0 — d n A -聚合酶依照 精於此項技術者習知之普通技巧將大腸桿菌1 i p A基因加以 放大。所用模板係大腸桿菌W311 0(ATCC 27325 )野生.型菌 株之染色體DNA。所用引物係具有下列順序、經磷硫酸酉旨 保護之募核苷酸lipASl及lipAS2 : 曰 lipASl :(順序識別號碼：3) 51- GAT CGA GGT CTC GAA TGA GTA AAC CCA TTG TGA TG -3!Page 12 200407430 V. Description of the invention (10) R a ~ Caprylic acid 'in order to prevent the false identification of the indicator strain in biological identification ~ The growth line is growing because (for example): In addition to R — α-lipoic acid, and Introduced glucose and acetic acid and succinic acid secreted by the producer (producer) strains, even if the growth of R-sulfur / caprylic acid producers is best using succinate as the sole carbon source. Bacteria are supplemented with the cell culture supernatant of the present invention; the growth of the indicator strain can then be followed to determine the lipoic acid content in the medium. The present invention will be further explained using the following experimental examples. The bacterial strain Escherichia coli used for the implementation of these routine examples "丨 ^ / 口 八" 一 丨 "^ 丨 丨 ^ was deposited under the Budapest Treaty under the number DSM 1 5 1 04 at the German Microbiology and Culture Cell Collection Company, Blanche Wig, Germany). Example 1: Construction of pASK-I ΒΑ3-1 i ρΑ vector (vector organism) A. 1 ipA gene amplification using polymerase chain reaction (p CR) using pw 0 — dn A-polymerization The enzyme amplifies the E. coli 1 ip A gene according to common techniques familiar to those skilled in the art. The template used is the chromosomal DNA of a wild-type strain of E. coli W311 0 (ATCC 27325). The primers used have the following sequence and are phosphate Nucleotides lipASl and lipAS2 protected by sulphuric acid: lipASl: (sequence identification number: 3) 51- GAT CGA GGT CTC GAA TGA GTA AAC CCA TTG TGA TG -3!
Bsal 1 ipAS2 :(順序識別號碼：4) 5*- GAT CGA GGT CTC GGC GCT CTT AAC TTC CAT CCC TTT CG -3丨 Bsal 隨後藉助於QI Aprep自轉（旋）小規模製備套具（奇亞_Bsal 1 ipAS2: (sequence identification number: 4) 5 *-GAT CGA GGT CTC GGC GCT CTT AAC TTC CAT CCC TTT CG -3 丨 Bsal then uses the QI Aprep to rotate (spin) a small-scale preparation kit (Chia_
200407430 五、發明說明（11) 公司、希爾頓，德國）之MA吸附柱依照製造商之說明將 内所製約1仟驗基之DNA碎片加以純化。 Β· 將1 ipA基因選殖入PASK-ΙΒΑ3載體内 _纟空由該等引物將Bsa I限制核酸内切酶之裂解（卵裂）部 t ^糖苷酸内強調之識別順序）引進該％1^碎片内。在製 ^。所指不之條件下藉B sa丨限制核酸内切酶將經純化之 卒#片加以裂解（卵裂），隨後於一瓊脂糖凝膠上加以分 ; = ;:GENE咖套具(BI0 101公司，拉荷拉，加州) “、、衣化商之說明從該瓊脂糖凝膠分離出來。 選殖及表現載體pASK-IBA3(IBA，生物分析研究所， ，德國）含有各種基因元素，該等基因元素容許一 土因之控制表現。此乃一具有複製起點，衍生自質體 P制且之高複製載體。該經選殖之基因受到tet阻遏物之壓 tae ΐτ藉（無水）四環素加以誘導。該載體亦更含有Strep_ 順序，該順序係直接結合選殖過程中經選殖基因之 ，哕*馬=丨如此所得經結合之基因現在可編碼一蛋白質 •川^ ^白負係*Strep_tag 11之胺基酸順序（SAWSHPQFEK 承員，識別號碼：5)實施C-終端伸展者。藉助於此標記可 二=°亥蛋白質之簡單親和純化作用，蓋因該StreP_tag n 职f結合作用中介給鏈抗生素蛋白柱。 在製造商所指示之條件下利用Ec〇31丨限制酶（BsaI等 =匆）裂解後pASK - IBA3載體而將1 ipA基因加以選殖，隨後 ，驗性碟酸酶處理將5，端去磷酸化，再藉助於GENECLEAN ^ 如同P C R碎片將該載體純化。依照製造商之說明並利200407430 V. Description of Invention (11) Company, Hilton, Germany) The MA adsorption column according to the manufacturer's instructions was used to purify the constrained 1 genomic DNA fragments. Β · 1 ipA gene was cloned into the PASK-ΙΒΑ3 vector. 纟 emptiness introduced these primers to the cleavage (cleavage) part of the Bsa I restriction endonuclease to introduce the recognition sequence in ^ glycoside. ^ Inside the debris. In Production ^. Under the conditions indicated, the purified puff # pieces were lysed (cleavage) by B sa 丨 restriction endonuclease, and then divided on an agarose gel; =;: GENE coffee kit (BI0 101 Company, La Jolla, California) "The instructions of the clothing manufacturer were separated from this agarose gel. The colony and expression vector pASK-IBA3 (IBA, Institute for Bioanalysis, Germany) contains various genetic elements, which Isogenic elements allow the control of a local cause. This is a highly replicative vector with origin of replication, derived from plastid P. The selected gene is pressured by the tet repressor tae ΐτ by (anhydrous) tetracycline This vector also contains the Strep_ sequence, which directly binds to the selected genes in the breeding process, 哕 * 马 = 丨 The resulting combined gene can now encode a protein • Chuan ^^ 白白 系 * Strep_tag 11 amino acid sequence (SAWSHPQFEK, identification number: 5) implement C-terminal stretcher. With this tag, two = simple affinity purification of the protein, Gein, the StreP_tag n binding function intermediary to Streptavidin protein Under the conditions indicated by the manufacturer, the 1 ipA gene was cloned with the pASK-IBA3 vector after cleavage by Ec31 丨 restriction enzyme (BsaI, etc.), and then, the 5′-end was removed by the test disciase treatment. Phosphorylated and the vector purified by means of GENECLEAN ^ like PCR fragments. Follow the manufacturer's instructions and benefit
第14頁 200407430 五、發明說明（12) 用T4 DNA連接酶使PCR碎片與經裂解及經去磷酸化之载體 連接。以精於此技術人員習知之方式藉助於電穿透作用利 用連接混合物將大腸桿菌DH 5 α細胞加以轉化。將該轉化 混合物塗敷在LB -氨苄青黴素瓊脂板上（丨〇公克/公升腺，5 公克/公升酵母萃取液，公克/公升NaC1，15公克/公升 f脂’ 1 0 0毫克/公升氨苄青黴素）並在3 71溫度下保溫過 夜。 ° 藉助於Q I Aprep自轉小規模製備套具（奇亞根公司，希 爾頓，德國）分離出該等質體之後，藉限制分析將預期 化物鑑別出來。 、如此製得之？人31(-18人3-1丨1^質體（第二圖）含有如以上 所述、、’Q &在忒lipA基因最後密碼子上之strep_tag順序。 但，該C-終端伸展與本發明無關，蓋因其對丨丨#蛋白質之 活性無重大影#，因此對本發明宿主生物之R…硫辛酸 之生產功能亦無重大影響。 實驗例2 : R- α -硫辛酸產體之製備 藉助於電穿透作用將實驗例丨内所述之pAsK一丨— 轉化為大腸桿菌W3110，於含有1〇〇毫克/公升氨〒青 黴素之LB瓊脂板上選擇之後’將該質體自轉化物之一中再 分離=來’用限制核酸内切酶加以裂解並加以檢查核對。 對照質體pASK-IBA3則以類似方式加以處理。 實驗例3 : R - α -硫辛酸之發酵製造 R - α -硫辛酸之發酵製造伤刹 厂Λ ό 知呀衣以係利用菌株W3110/pASK-ΙΒΑ3- ΙιρΑ 〇為作比車交，含有dASK - TRAQ所 ^ iBA3質體之菌株W3110係在完Page 14 200407430 V. Description of the invention (12) T4 DNA ligase is used to link the PCR fragments to the lysed and dephosphorylated vector. E. coli DH5? Cells were transformed in a manner familiar to those skilled in the art by means of electropenetration using a ligation mixture. The conversion mixture was coated on an LB-ampicillin agar plate (0 g / L gland, 5 g / L yeast extract, g / L NaC1, 15 g / L f lipid '100 mg / L ampicillin ) And incubate at 37 1 overnight. ° With the help of Q I Aprep rotation small-scale preparation kits (Kiagen, Hilden, Germany), the plastids were separated and the expected compounds were identified by restriction analysis. , So made? Human 31 (-18 human 3-1 丨 1 ^ plastids (second picture) contains the strep_tag sequence on the last codon of the lipA gene as described above. However, the C-terminal extension and The present invention is irrelevant, because it has no significant effect on the #protein activity, and therefore has no significant effect on the production function of R ... lipoic acid of the host organism of the present invention. Experimental Example 2: R-α-lipoic acid producing body The pAsK- described in Experimental Example 丨 was transformed into E. coli W3110 by electro-penetration, and the plastid was transformed from the transformant after selection on an LB agar plate containing 100 mg / liter ampicillin. Re-isolation in one of them = come 'cleavage with restriction endonucleases and check. The control plastid pASK-IBA3 is treated in a similar way. Experimental Example 3: R-α-Fermentation by Lipoic Acid -Lipoic acid fermentation to produce wound brake plant Λ ό Zhiyayi uses strain W3110 / pASK-ΙΒΑ3- ΙιρΑ 〇 as a comparison vehicle, containing strain dASK-TRAQ ^ iBA3 plastid strain W3110 is in the end
200407430 五、發明說明（13) 全相同之條件下培養者。 作為產體培養之命· 土立 吉/八井气主主1 I 養囷種，f先將5毫升含有1〇〇毫 β液體培養基與各個菌株實施保溫 亚於振盪裔上，以每分鐘16〇轉之轉速，在3?1^产 下保溫i6小2。藉離心作用將該等細胞採集出來，並7對 f f ::食鹽水(°.9% Na⑴清洗兩次。最後用如此 衣付之、.、田胞保溫i 5毫升BS培養基（7公克/公升Κ2Ηρ〇4 ; 3公 克^ Γ=4 U t克/公升（NH4)2S〇4; 〇.1公克/公升心叫 X 2/ 么克/公升Na3檸檬酸X 3ϋ2Ο;0.2%酸水解 酷蛋白（無維生素）；13.5公克/公升-號㈣χ 6η文2〇'解 酸度值用HC1调整至6. 8)其中更含有1〇〇毫升/公 黴素，以！ ：1〇〇比例。於—振盡器上，以每 轉速’⑽t溫度下將料產體培養物加以保溫48小轉時之 保溫約4小時之後添加〇. 2毫克/公升無水四環素以护 辛酸-合成酶基因之表現。於24小時及48小時之後 2 樣品並f離心作用將該等細胞自培養基内取出。藉助於出習 知之濁度什生物檢定（赫伯特及蓋斯特，197〇 18A，269至272)將其中所含R—㈠泉辛酸加以量化1子 :示係在該特殊培養物幻青液内戶…之含中 罝·200407430 V. Description of invention (13) Trainees under the same conditions. As the lifeblood of culture, Turichi / Hachii is the main breeder of I I, and first, 5 ml of a liquid medium containing 100 milli betas is incubated with each strain on the shaker at 16 per minute. With a rotation speed of 〇rpm, the heat preservation i6 is smaller than 2 at 3? The cells were collected by centrifugation and washed twice with 7 pairs of ff :: saline (° .9% Na⑴). Finally, they were coated with 5 ml BS medium (7 g / L) Κ2Ηρ〇4; 3g ^ Γ = 4 U tg / liter (NH4) 2S〇4; 0.1g / liter heart is called X 2 / μg / liter Na3 citric acid X 3ϋ20; 0.2% acid hydrolyzed cool protein ( No vitamins); 13.5 g / litre-No. ㈣χ 6η 文 20 'deacidification value adjusted with HC1 to 6.8) which contains 100 ml / commonamycin, with a ratio of :: 100. Yu-Zhen On the whole device, the culture of the production body was maintained at a temperature of 48 ° C per rotation speed for about 4 hours, and then 0.2 mg / liter of anhydrous tetracycline was added to protect the performance of the caprylic acid-synthetase gene. At 24 After 2 hours and 48 hours, the samples were removed from the culture medium by centrifugation. The cells were removed by means of the conventional turbidity bioassay (Herbert and Geist, 19701A, 269 to 272). Contains R-Yanquan octanoic acid to quantify 1 child: it is shown in the special culture of the magic green liquid house ...
第16頁 200407430 五、發明說明 (14) 1 · 順序表 <110> 電化工業聯合公司 5 <12〇> 分泌R-α -硫辛酸之绰跑及該R -a -硫辛酸之發酵製造方法 ^130> C010207 / P 10 <140> <141> <!〇0> 5 15 <170> Patentln Ver. 2.0 <210> <211> 1 9ββ 20 <212> DNA <213> 大腸桿菌 <220> <221> CDS 25 <222> (1) . · (963) <300> <301> 雷德 ，凱林. .E . 30 <302> 克羅南， 約翰E· 硫辛酸在大腸桿菌中之代謝作用 ：lipA 及lipB基因之順序測定及功能特性描述 <303>麵菌學報 <304> 175 35 <305> 5 <30β> 1325-1336 <307> 1993 <400> 1 40 atg agt aaa GCC att gtg atg gaa cgc ggt gt:t aaa tae t=gc gat gcc 48 Met Ser Lys Pro lie Val Met Glu Arg Gly Val Lys Tyr Arg Asp Ala 1 5 10 15 gat aag atg gcc ett ate ccg gtt aaa aac gtg gca aca gag cgc gaa 96 45 Asp Lys Met Ala Leu lie Pro Val Lys A^n Val Ala Thr Cilu 'Arg Glu 20 25 30 gcc ctg ctg cgc aag ccg gaa tgg atg aaa ate aag ett cca geg gac 144 Ala Leu Leu Arg Lys Pro Giu Trp Met Lys lie Lys Leu Pro Ala Asp 50 35 40 45 tct aca cgt ate cag ggc ate aaa gcc gca atg cgc aaa aat ggc ctg 192 Sar Thr Arg lie Gin Gly lie Lys Ala Ala Met Arg Lys Asn Gly Leu 50 55 60 55 cat tct gtc tgc gag gaa gcc tcc tgc cct aac ctg geg gaa tgc ttc 240 His Ser Vai Cys Gin Gla Ala Ser Cys Pro Asn Leu Ala Glu Cys Pine 65 70 75 80 60 aac cac ggc aca gca aeg tut atg ate etc ggc get att tgt acc cgc 238Page 16 200407430 V. Description of the invention (14) 1 · Sequence table < 110 > Dianhe Industry Co., Ltd. 5 < 12〇 > Secretion of R-α-lipoic acid and fermentation of the R-a-lipoic acid Manufacturing method ^ 130 > C010207 / P 10 < 140 > < 141 > <! 〇0 > 5 15 < 170 > Patentln Ver. 2.0 < 210 > < 211 > 1 9ββ 20 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS 25 < 222 > (1). · (963) < 300 > < 301 > Ryder, Kailin. .E. 30 < 302 > g Luo Nan, John E. Lipoic Acid Metabolism in Escherichia coli: Sequence Determination and Functional Characterization of LipA and LipB Genes < 303 > Journal of Polymycology < 304 > 175 35 < 305 > 5 < 30β > 1325 -1336 < 307 > 1993 < 400 > 1 40 atg agt a aa GCC att gtg atg gaa cgc ggt gt: t aaa tae t = gc gat gcc 48 Met Ser Lys Pro lie Val Met Glu Arg Gly Val Lys Tyr Arg Asp Ala 1 5 10 15 gat aag atg gcc ett ate ccg gtt aaa aac gtg gca aca gag cgc gaa 96 45 Asp Lys Met Ala Leu lie Pro Val Lys A ^ n Val Ala Thr Cilu 'Arg Glu 20 25 30 gcc ctg ctg cgc aag ccg gaa tgg atg aaa ate aag ett cca geg gac 144 Ala Leu Leu Arg Lys Pro Giu Trp Met Lys lie Lys Leu Pro Ala Asp 50 35 40 45 tct aca cgt ate cag ggc ate aaa gcc gca atg cgc aaa aat ggc ctg 192 Sar Thr Arg lie Gin Gly lie Lys Ala Ala Met Arg Lys Asn Gly Leu 50 55 60 55 cat tct gtc tgc gag gaa gcc tcc tgc cct aac ctg geg gaa tgc ttc 240 His Ser Vai Cys Gin Gla Ala Ser Cys Pro Asn Leu Ala Glu Cys Pine 65 70 75 80 60 aac cac ggc aca gca aeg tut atg ate etc ggc get att tgt acc cgc 238
•第17頁 200407430 五、發明說明（15)• Page 17 200407430 V. Description of the invention (15)
Asn. His Gly Thir A丄s Thr ?π.0 M0c H lb Leu Glv Ala. He Cvs Thr 85 90 95 z 0. as gA t o c r c ? t a o c 1 1 gAi all t a tnv cr-o c r G p να 3 3 cgt tgt ccg etc tgt caz gtt gcc cac gg- cgc Arg Cys Pro Phe Cvs Asp Val Ala His Gly Arc 100 105 ^ — gcc aau gaa cca gtg aaa ctg geg cag acc att gcc gat atg geg czg 334Asn. His Gly Thir A 丄 s Thr? Π.0 M0c H lb Leu Glv Ala. He Cvs Thr 85 90 95 z 0. as gA tocrc? Taoc 1 1 gAi all ta tnv cr-o cr G p να 3 3 cgt tgt ccg etc tgt caz gtt gcc cac gg- cgc Arg Cys Pro Phe Cvs Asp Val Ala His Gly Arc 100 105 ^ — gcc aau gaa cca gtg aaa ctg geg cag acc att gcc gat atg geg czg 334
Ala Asn Glu Pro Val Lys Leu Ala Gin Thr lie Ala Aso Mst Aia Leu 10 115 120 125 cgt tat gtg gtt ate acc tcc gtt gac cgt gat gac ctg cgc gat ggc 432Ala Asn Glu Pro Val Lys Leu Ala Gin Thr lie Ala Aso Mst Aia Leu 10 115 120 125 cgt tat gtg gate ate acc tcc gtt gac cgt gat gac ctg cgc gat ggc 432
Arg Tyr Val Val lie Thr Ser Val Asp Arg Asp Asp Leu Arg Aso GIv 130 135 ' 140 " ggt gcc cag cac ttt geg gat tgc att: act gcc att egg gaa aaa age 480Arg Tyr Val Val lie Thr Ser Val Asp Arg Asp Asp Leu Arg Aso GIv 130 135 '140 " ggt gcc cag cac ttt geg gat tgc att: act gcc att egg gaa aaa age 480
Gly Ala Gin Kis Phe Ala Asp Cvs lie Thr Ala lie Aj：g Glu Lys Ser 145 150 155 160 20 ccg Pro caa ate aaaGly Ala Gin Kis Phe Ala Asp Cvs lie Thr Ala lie Aj: g Glu Lys Ser 145 150 155 160 20 ccg Pro caa ate aaa
att gaa act ctg gtg ccg gat ttc cgc ggt cgt atg lie Glu Thr Leu Val Pro Asp Phe Arg Gly Arg Met 165 170 175 528 gat cgt get ctg gat att ctg act gca aeg cca cca gat gtg ttc aac 576 25 Asp Arg Ala Leu Asp lis Leu Thr Ala Thr Pro Pro Asp Val Phe Asn 180 135 190 cat aac ctg gaa aac gta ccg cgt: att tac cgt cag gta egg cct ggt 624att gaa act ctg gtg ccg gat ttc cgc ggt cgt atg lie Glu Thr Leu Val Pro Asp Phe Arg Gly Arg Met 165 170 175 528 gat cgt get ctg gat att ctg act gca aeg cca cca gat gtg ttc aac 576 25 Asp Asp lis Leu Thr Ala Thr Pro Pro Asp Val Phe Asn 180 135 190 cat aac ctg gaa aac gta ccg cgt: att tac cgt cag gta egg cct ggt 624
His Asn Leu Glu Asn Val Pro Arg lie Tyr Arg Gin Val Arg Pro Giy 30 195 200 205 gca gat tac aac tgg teg ctg aag ctg ctg gaa cgc ttt aaa gaa geg 672 Ala Asp Tyr Asn Trp Ser Leu Lys Leu Leu Glu Arg Phe Lys Glu Ala 35 210 215 220 cat ccg gaa ate ccg acc aag tet ggt ctg atg gtg gga ctg ggt gaa 720 His Pro Glu He Pro Thr Lys Ser Gly Leu Met Val Gly Leu Gly Glu 225 230 235 240 40 acc aat gaa gaa att att gag gta atg cgc gac ctg cgc cgt cat ggt 7 63His Asn Leu Glu Asn Val Pro Arg lie Tyr Arg Gin Val Arg Pro Giy 30 195 200 205 gca gat tac aac tgg teg ctg aag ctg ctg gaa cgc ttt aaa gaa geg 672 Ala Asp Tyr Asn Trp Ser Leu Lys Leu Leu Glu Arg Pug Lys Glu Ala 35 210 215 220 cat ccg gaa ate ccg acc aag tet ggt ctg atg gtg gga ctg ggt gaa 720 His Pro Glu He Pro Thr Lys Ser Gly Leu Met Val Gly Leu Gly Glu 225 230 235 240 40 acc aat gaa gaa att att gag gta atg cgc gac ctg cgc cgt cat ggt 7 63
Thr Asn Glu Glu lie lie Glu Val Met Arg Asp Leu Arg Arg Kis Gly 245 250 255 gt:g aeg atg tta aeg ctg ggg caa tat ttg cag cca age cgc cat cac 8lo 45 Val Thr Met Leu Thr Leu Gly Gin Tyr Leu Gla Pro Ser Arg 'His His 260 265 270 ctg ccg gtt caa cgt tac gtt age ccg gat gag ttc gac gaa atg aaa 3 64Thr Asn Glu Glu Glu lie lie Val Glu Met Arg Asp Leu Arg Arg Kis Gly 245 250 255 gt: g aeg atg tta aeg ctg ggg caa tat ttg cag cca age cgc cat cac 8lo 45 Val Thr Met Leu Thr Leu Gly Gin Tyr Leu Gla Pro Ser Arg 'His His 260 265 270 ctg ccg gtt caa cgt tac gtt age ccg gat gag ttc gac gaa atg aaa 3 64
Leu Pro Val Gla Arg Tyr Val Ser Pro Asp Glu Phe Asp Glu Met Lys 50 275 230 · 285 55 ccc Ala gaa Glu 290 geg Ala ctg Leu geg iUa atg Met ggc Gly 295 tt- Phe acc Thr cat His get Ala gca Ala 300 tgc Cys ggt Gly ccg Pro ttt Phe 912 Val 305 cgc Arg tet Ser tet Ser tac Tyr cac His 310 gee Ala ga 二 As? ttg Leu cag Gin geg Ala 315 aaa Lys ggg Gly atg Met gaa Glu Val 320 960 60 aag taa 966 第18頁 200407430 五、發明說明（16)Leu Pro Val Gla Arg Tyr Val Ser Pro Asp Glu Phe Asp Glu Met Lys 50 275 230 · 285 55 ccc Ala gaa Glu 290 geg Ala ctg Leu geg iUa atg Met ggc Gly 295 tt- Phe acc Thr cat His get Ala gca Ala 300 tgc Cys ggt Gly ccg Pro ttt Phe 912 Val 305 cgc Arg tet Ser tet Ser tac Tyr cac His 310 gee Ala ga di As? ttg Leu cag Gin geg Ala 315 aaa Lys ggg Gly atg Met gaa Glu Val 320 960 60 aag taa 966 Page 18, 200407430 V. Description of the invention (16)
Lys <210> 2 5 <211> 321 <212> PRT<2L3>大腸桿菌 <400> 2 i〇 Met Ser Lys Pro lie Val Met GIu Arg Giy Vai Lys Tyr Arg Aso Ala 1 5 10 15 15Lys < 210 > 2 5 < 211 > 321 < 212 > PRT < 2L3 > E. coli < 400 > 2 i〇 Met Ser Lys Pro lie Val Met GIu Arg Giy Vai Lys Tyr Arg Aso Ala 1 5 10 15 15
Asp Lys Met Ala Leu lie Pro Val Lys Asn Val Ala Thr Giu Arg Glu 20 25 30 A丄a Leu Leu Arg Lys Pro Glu Trp Met Lys lie Lys Leu Pro Ala Asp 35 40 45Asp Lys Met Ala Leu lie Pro Val Lys Asn Val Ala Thr Giu Arg Glu 20 25 30 A 丄 a Leu Leu Arg Lys Pro Glu Trp Met Lys lie Lys Leu Pro Ala Asp 35 40 45
Ser Thr Arg lie Gin Gly lie Lys Ala Ala Met Arg Lys Asn Giy Leu 20 50 55 60 His Ser Val Cys Glu Glu Ala Ser Cys Pro Asn Leu Ala Glu Cys Phe 65 70 75 80 25 Asn His Giy Thr Ala Thr Phe Met lie Leu Giy Ala lie Cys Thr Arg 85 90 95 30Ser Thr Arg lie Gin Gly lie Lys Ala Ala Met Arg Lys Asn Giy Leu 20 50 55 60 His Ser Val Cys Glu Glu Ala Ser Cys Pro Asn Leu Ala Glu Cys Phe 65 70 75 80 25 Asn His Giy Thr Ala Thr Phe Met lie Leu Giy Ala lie Cys Thr Arg 85 90 95 30
Arg Cys Pro Phe Cys Asp Val Ala His Giy Arg Pro Val Ala Pro Asp ^ 100 1Q5 110 Ala Asn Glu Pro Val Lys Leu Ala Gin Thr lie Ala Asp Met Ala I*eu 115 120 125 .rArg Cys Pro Phe Cys Asp Val Ala His Giy Arg Pro Val Ala Pro Asp ^ 100 1Q5 110 Ala Asn Glu Pro Val Lys Leu Ala Gin Thr lie Ala Asp Met Ala I * eu 115 120 125 .r
Arg Tyr Val Val 工le Thr. Ser Val Asp Arg Aso Aso Leu Arg Aso Gly 35 130 135 140 " Giy Ala Gin His Phe Ala Asp Cys Ila Thr Ala lie Arg Glu Lys Ser 145 150 155 160 40 Pro Gin lie Lys lie Glu Thr Leu Val Pro Asp Phe Arg Gly Arg Met 165 170 ' 175 45Arg Tyr Val Val Gong Thr. Ser Val Asp Arg Aso Aso Leu Arg Aso Gly 35 130 135 140 " Giy Ala Gin His Phe Ala Asp Cys Ila Thr Ala lie Arg Glu Lys Ser 145 150 155 160 40 Pro Gin lie Lys lie Glu Thr Leu Val Pro Asp Phe Arg Gly Arg Met 165 170 '175 45
Asp Arg Ala Leu Asp lie Leu Thr Ala Thr Pro Pro Asp Vai Phe Asn 180 135 190 His Asn Lea Gla Asn Val Pro Arg lie Tyr Arg'Gin Val Arg Pro Giy 195 200 205Asp Arg Ala Leu Asp lie Leu Thr Ala Thr Pro Pro Asp Vai Phe Asn 180 135 190 His Asn Lea Gla Asn Val Pro Arg lie Tyr Arg'Gin Val Arg Pro Giy 195 200 205
Ala Asp Tyr Asa Trp Ser Leu Lys Leu Leu Giu. Arg Phe Lys Glu Ala 50 210 ' 215 220Ala Asp Tyr Asa Trp Ser Leu Lys Leu Leu Giu. Arg Phe Lys Glu Ala 50 210 '215 220
His Pro Glu lie Pro Thr Lys Ser Gly Leu Met Vai 225 230 235His Pro Glu lie Pro Thr Lys Ser Gly Leu Met Vai 225 230 235
Gly Giu 240 55 Thr Asa Glu Gla lie IIs Giu Val Met Arg Asp Leu Arg Arg His Gly 245 250 ~ 255Gly Giu 240 55 Thr Asa Glu Gla lie IIs Giu Val Met Arg Asp Leu Arg Arg His Gly 245 250 ~ 255
Val Thr Met Leu Thr Leu Giy Gin Tyr Leu Gin. Pro Ser Arg Kis His 260 265 270 60Val Thr Met Leu Thr Leu Giy Gin Tyr Leu Gin. Pro Ser Arg Kis His 260 265 270 60
Hi 第19頁 200407430 五、發明說明（17)Hi Page 19 200407430 V. Description of the invention (17)
Leu Pro Val Gla Arg Tyr Val Ser Pro As? GIu Phe Aso Glu iMet Lvs 275 230 285 ** _ Ala Glu Ala Leu Ala Met Giy Phs Thr His Ala Ala Cvs Gly Pro Phe 5 290 295 300 一Leu Pro Val Gla Arg Tyr Val Ser Pro As? GIu Phe Aso Glu iMet Lvs 275 230 285 ** _ Ala Glu Ala Leu Ala Met Giy Phs Thr His Ala Ala Cvs Gly Pro Phe 5 290 295 300 1
Val Arg Ser Ser Tyr His Ala As? Leu Gin Ala Lvs Glv Met Glu Val 305 310 315 * 320 10 Lys <210> 3 15 <211> 35 <212> DMA <2U>人造順序 <220> 20 <223>人造順序之說明·•寡核苷酸lipASl <400> 3 gatcgaggtc tcgaatgagt aaacccattg tgatg 35 25Val Arg Ser Ser Tyr His Ala As? Leu Gin Ala Lvs Glv Met Glu Val 305 310 315 * 320 10 Lys < 210 > 3 15 < 211 > 35 < 212 > DMA < 2U > Artificial Sequences < 220 > 20 < 223 > Explanation of the artificial sequence • oligonucleotidelipASl < 400 > 3 gatcgaggtc tcgaatgagt aaacccattg tgatg 35 25
<210> 4 <211> 38 <212> DNA 30 <213〉人造順序 <220> .· <223>人造順序之說明：寡核苷酸lipAS2 35 <400> 4 * gatcgaggtc tcggcgctct taacttccat ccctttcg 38 40 <210> 5 <211> 10 <212> PRT <213>人造順序 45 <220> * <223〉人造順序之說明：strep-tag E順序 <400> 5 50 Ser Ala Trp Ser His Pro Gin Phe Glu Lys 15 10 第20頁 200407430 圖式簡單說明 第一圖：R-〇;-硫辛酸在大腸桿菌中之合成 第一圖：PASK-IBA3-1 ipA 載體< 210 > 4 < 211 > 38 < 212 > DNA 30 < 213> Artificial sequence < 220 >. < 223 > Explanation of artificial sequence: oligonucleotide lipAS2 35 < 400 > 4 * gatcgaggtc tcggcgctct taacttccat ccctttcg 38 40 < 210 > 5 < 211 > 10 < 212 > PRT < 213 > artificial order 45 < 220 > * < 223> Explanation of artificial order: strep-tag E-order < 400 > 5 50 Ser Ala Trp Ser His Pro Gin Phe Glu Lys 15 10 Page 20 200407430 Simple illustration of the first picture: Synthesis of R-〇; -lipoic acid in E. coli First picture: PASK-IBA3-1 ipA vector
Claims (1)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10235270A DE10235270A1 (en) | 2002-08-01 | 2002-08-01 | Cells that secrete R-alpha-lipoic acid and process for the fermentative production of R-alpha-lipoic acid |
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| Publication Number | Publication Date |
|---|---|
| TW200407430A true TW200407430A (en) | 2004-05-16 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW092121056A TW200407430A (en) | 2002-08-01 | 2003-07-31 | Cells secreting R-α- lipoic acid and method for fermentative production of said R-α- lipoic acid |
Country Status (4)
| Country | Link |
|---|---|
| AU (1) | AU2003260329A1 (en) |
| DE (1) | DE10235270A1 (en) |
| TW (1) | TW200407430A (en) |
| WO (1) | WO2004013314A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10258127A1 (en) * | 2002-12-12 | 2004-07-08 | Consortium für elektrochemische Industrie GmbH | Process for the fermentative production of R-α-lipoic acid |
| WO2009091582A1 (en) * | 2008-01-17 | 2009-07-23 | Indigene Pharmaceuticals, Inc. | PRODUCTION OF R-α-LIPOIC ACID BY FERMENTATION USING GENETICALLY ENGINEERED MICROORGANISMS |
| CN111206035B (en) * | 2020-02-17 | 2021-12-28 | 河南科技学院 | Gene and method for regulating senescence process of target plant leaves and application of gene and method to cotton crops |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20040235123A1 (en) * | 2001-04-20 | 2004-11-25 | Liao Hans H | Production of alpha-lipoic acid |
-
2002
- 2002-08-01 DE DE10235270A patent/DE10235270A1/en not_active Ceased
-
2003
- 2003-07-24 WO PCT/EP2003/008178 patent/WO2004013314A1/en not_active Application Discontinuation
- 2003-07-24 AU AU2003260329A patent/AU2003260329A1/en not_active Abandoned
- 2003-07-31 TW TW092121056A patent/TW200407430A/en unknown
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| Publication number | Publication date |
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| DE10235270A1 (en) | 2004-02-12 |
| AU2003260329A1 (en) | 2004-02-23 |
| WO2004013314A1 (en) | 2004-02-12 |
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