TW200540269A - Novel transformant - Google Patents
Novel transformant Download PDFInfo
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- TW200540269A TW200540269A TW094111212A TW94111212A TW200540269A TW 200540269 A TW200540269 A TW 200540269A TW 094111212 A TW094111212 A TW 094111212A TW 94111212 A TW94111212 A TW 94111212A TW 200540269 A TW200540269 A TW 200540269A
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- Taiwan
- Prior art keywords
- leu
- polyester
- composition
- ala
- plastid
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Description
200540269 九、發明說明： 【發明所屬之技術領域】 本發明係關於高3HH組成型聚酯之生產用新穎質體，及 包含該質體之轉形體，以及使用該質體之高3HH組成型聚 • 酯之製造方法。 【先前技術】 迄今已知許多微生物在菌體内積蓄聚酯以做為能量貯藏 φ 物為。其之代表例，如為3-羥基丁酸（以下簡稱為3HB)均聚 物之聚3-羥基丁酸（以下簡稱為P(3HB))，其最早係於1925 年在巨大芽孢桿菌（Bacillus megaterium)中發現。p(3HB)為 熱塑性高分子，由於可在自然環境中被生物分解，為有益 於環保之塑膠而受人注目。然而，p(3HB)由於結晶性高， 具有硬且脆之性質，在實用方面其應用範圍受到限制。因 此’其性質之改良乃成為研究之目的。 其中，曾揭示包含3-羥基丁酸（3HB)與3_羥基纈草酸（3HV) • 之共聚合體（以下簡稱為P(3HB-CO-3HV))之製造方法（例如 參照專利文獻1及專利文獻2)。該p(3HB_co-3HV)與P(3HB) 相較’富於柔軟性，可應用於廣泛之用途。然而，實際上 就P(3HB-CO-3HV)而言，即使增加3HV之莫耳比，隨之產生 之物性變化仍嫌不足，尤其無法提高在薄膜加工等方面所 要求之柔軟性，而只能用於洗髮精瓶或用後即丟型剃刀之 、 把手等硬質成形體之領域。 近年，研究有關3HB與3-羥基己酸（以下簡稱為3ηη)之二 成分共聚合聚酯（以下簡稱為p(3HB-co-3HH))及其製造方 100136.doc 200540269 法（參照專利文獻3及專利文獻4)。此等報告之 P(3HB-CO-3HH)之製造方法，係使用從土壤單離之豚鼠產氣 單胞桿菌（Ae romonas caviae)使油酸等脂肪酸或橄欖油等油 脂發酵而生產者。又，亦對有關P(3HB-co-3HH)之性質進行 研究（參照非專利文獻1)。在此報告中，以碳數為12個以上 之脂肪酸做為唯一碳源培養豚鼠產氣單胞桿菌，發酵生產 3HH組成為η至19莫耳％之p(3hb_CO-3HH)。該 P(3HB-CO-3HH)隨著3HH組成之增加，從P(3HB)之硬且脆之 性質逐漸呈現柔軟之性質，明顯地提高P(3HB-C〇-3HH)之柔 軟性。亦即，P(3HB_C0-3HH)藉由改變3HH組成，具有可應 用於硬質聚合物乃至軟質聚合物之寬廣物性，因而可期待 將其應用於如電視機外殼等要求硬度者至如薄膜等要求柔 軟性者之寬廣領域。然而，由於本製造方法菌體生產量為 4g/L’聚合物含量為3〇%且聚合物生產性低，仍稱不上係可 邁向實用化之生產方法，因此仍待探索可實用化且生產性 更高之方法。 以P(3HB-co-3HH)之工業生產為目標進行研究改進。在使 用耆水氣單胞桿菌（Aer〇m〇nas hydrophila)之培養中，以油 酉文做為奴源進行43小時之流加（fed_batch)培養，可生產菌體 里為95.7 g/L，聚合物含量為45·2%且3HH組成為17%之 4(3HB-CO-3HH)(參照非專利文獻2)。又，將嗜水氣單胞桿 菌使用㈣糖及月桂酸做為碳源進行培養，達利體量為 老gL聚合物含1為5〇%(參照非專利文獻。然而，由於 嗜水氣單胞桿菌對人類有致病性（參照非專利文獻4)，因此 100136.doc 200540269 非為適a工業生產之品種。χ，由於在此等培養生產中使 用高價格之碳源，從製造成本之觀點而言，亦必須尋求利 用廉價碳源者。 φ 因此，乃以採用安全宿主之生產及生產性之提高為目標 進行研九改進。從嗜水氣單胞桿菌進行聚羥基烧酸（PHa) 合成酵素基因之選殖（參照專利文獻5及非專利文獻5)。使用 等本基因導入真養雷爾氏痛eUtr〇pha，舊名真養 馨產鹼杯菌（Alcaligenes eutr〇phus))而得之轉形體生產 POHB-codHH)，結果菌體生產量為4 g/L，聚合物含量為 3 0/。。再者，將本轉形體使用植物油脂做為碳源進行培養， 結果菌體含量達拉斯4g/L，聚合物含量8〇%(參照非專利文 獻）：、彳而以廉價植物油脂做為碳源者之聚合物生產性 低，又其3HH組成為5%，具有硬且脆之物性。 又，以果糖做為碳源雖可構築能生產p(3HB_c〇_3HH)之真 養雷爾氏菌，然而本菌株之聚合物生產性低，不適於實際 • 生產（參照非專利文獻7)。 亦可構築以大腸菌做為宿主之p(3HB_c〇-3HH)生產株。構 築將氣單胞桿菌（Aeromonas)屬之PHA合成酵素基因等及真 養雷爾氏菌之NADP-乙醯乙醯基c〇-A還原酵素基因導入大 腸菌之株。以十二烷做為碳源將相同大腸菌培養4〇·8小時 之^果’菌體量為79 g/L，聚合物含量為27.2%，3ΗΗ組成 -為10·8°/。（參照非專利文獻8)。 亦可構築將豚鼠產氣單胞桿菌之PHA合成酵素基因、乙 醯基CoA水合酶及醯基coA脫氫酵素基因導入之大腸菌。 100136.doc 200540269 若將相同大腸菌在含有月桂酸之培養基中培養，聚合物含 $為16°/。，3HH組成為16%(參照非專利文獻9)。使用此等大 腸菌生產性低，難以適用工業化之生產。 另一方面，以提高P(3HB-co-3HH)之生產性及控制3HH組 成為目標，進行PHA合成酵素之人為改變（非專利文獻1〇及 非專利文獻11)。從豚鼠產氣單胞桿菌而來之PHA合成酵素 變異體中，第149號胺基酸天冬醯胺酸被絲胺酸取代之變異 體酵素及第171號胺基酸天冬醯胺酸被甘胺酸取代之變異 體酵素，顯示可提高大腸菌内PHA合成酵素活性及3HH組 成；又，曾報導第5 1 8號胺基酸苯丙胺酸被異白胺酸取代之 變異體酵素及第214號胺基酸纟領草胺酸被甘胺酸取代之變 異體酵素顯不可長;向大腸痛内PH A合成酵素活性及聚合物 含量。然而，由於其等使用特殊大腸菌做為宿主聚合物含 量仍低，要利用此等變異體酵素之特徵進行工業化生產仍 需要進一步之改良。 T. Fukui等以真養單胞菌做為宿主生產p(3HB-c〇-3HH)之 PHA合成酵素表現質體，係將聚酯合成酵素基因及D_烯醇
Co-A 水合酶（hydratase)基因等導入 pJRD215 (ATCC 37533) 而成之PJRDEE32及PJRDEE32dl3等（參照專利文獻5)。本菌 株之菌體含量雖低，只有4 g/L，然而藉由以植物油脂做為 石厌源改善該鹵株之培養條件，可提高菌體含量至4 5 g/L，聚 合物含量至62 _ 5 %，3 HH組成至8 · 1 % ;亦曾研究依照培養條 件生產高3HH組成之P(3HB-co-3HH)之方法（參照專利文獻 6) 〇 100136.doc 200540269 又，亦揭示控制P(3HB_co-3HH)物性之方法（參照專利文 獻6)。藉由使用至少2種碳數不同之油脂及/或脂肪酸做為碳 源，可以生產3HH組成為1至40莫耳％2P(3HB_c〇-3HH)， 因而可生產具有各種物性之P(3HB-C0_3HH)。然而，在該製 造方法中，為控制3HH組成，必須添加較高價之己酸或辛 酉欠4，又由於兩》農度之己酸呈現細胞毒性，結果使菌體生 產性降低。再者，由於添加多成分之碳源，生產設備可能 變得複雜及昂貴。 [專利文獻1]特開昭57-150393號公報 [專利文獻2]特開昭59-220192號公報 [專利文獻3]特開平5-93049號公報 [專利文獻4]特開昭7-265065號公報 [專利文獻5]特開昭10-108682號公報 [專利文獻6]特開昭2001-340078號公報 [非專利文獻 1] Y. Doi，S. Kitamura，H. Abe，Macromolecules 28, 4822-4823 (1995) [非專利文獻 2] Biotechnology and Bioengineering，vol 67, 240 (2000) [非專利文獻 3] Appl· Microbiol. Biotechnol·，vol 57，50 (2001) [非專利文獻4]日本國立感染症研究所，病原體等安全 規定，別表1附表1 (1999) [非專利文獻 5] T· Fukui，Y. Doi，J. Bacteriol，179，15, 4821-4830 (1997) 100136.doc 200540269 [非專利文獻 6] T. Fukui 等，Appl. Microbiol. Biotechnol· 49, 333 (1998) [非專利文獻 7] T· Fukui 等，Biomolecules，vol 3，618 (2002) [非專利文獻 8] S· Park等，Biomacromolecules，vol 2，248 (2001) [非專利文獻 9] X· Lu等，FEMS Microbiology Letters，vol 221，97 (2003) [非專利文獻 10] T. Kichise等，八卩卩1.£1^1*〇11.]^(^〇1^〇1· 68, 2411-2419 (2002) [非專利文獻 11] A· Amara等，App. Microbiol. Biotechnol·， vol 59, 477 (2002) 【發明内容】 發明之揭示 發明欲解決之課題 從安全性、高生產性及可利用廉價碳源之觀點而言，可 以期待真養雷爾氏_ (Ralst〇nia eutr〇pha)為工業生產 P(3HB-co-3HH)之最適當宿主。然而，由於與低3HH組成型 P(3HB-C0-3HH)生產系統相較，利用柔軟性質而適於加工成 薄膜等之高3HH組成型p(3HB-co-3HH)之安全且廉價之生 產系統尚未確立，因此仍期待開發其之高生產系統。 解決此課題之手段 本發:者為解決上述課題進行專心檢討，構築含有從豚 鼠產氣單胞桿菌而來之phaC新穎衍生物基因之新顆聚醋合 100136.doc 10 200540269 成酵素表現質體。製作將該表現質體導入真養雷爾氏菌等 宿主而成之轉形體，並以廉價之植物油脂做為唯一碳源進 行培養，結果發現能以高生產性生產3HH組成為12莫耳％以 上之 P(3HB-co-3HH)。 亦即，本發明係關於能製造在加工成薄膜等加工性方面 優良之高3HH組成型P(3HB-co-3HH)之聚酯合成酵素表現 質體，含有該質體之具有高3HH組成型聚酯合成能力之轉 形體，及使用該質體之高3HH組成型聚酯之安全且廉價之 製造方法。 總之，本發明係關於高3HH組成型聚酯合成酵素表現質 體，即 PHB-4/pJRDdTc+149NS171DG (FERM BP-10259 及 FIRDI BCRC 940479)所包含之聚酉旨合成酵素表現質體（I)， 將上述表現質體小型化之聚酯合成酵素表現質體（II)，藉由 上述高3HH組成型聚酯合成酵素表現質體轉形之轉形體及
PHB-4/pJRDdTc+149NS171DG (FERM BP-1 0259 及 FIRDI BCRC 940479)，及使用轉形體之聚酯製造方法。上述製造 方法以使用上述轉形體之高3HH組成型聚酯之安全且廉價 之製造方法為較佳，其中之聚酯為下式（1) Γ U 、 Γ η u 飞 Γ ch3 Ί r c3h7 η Η — 1 —O C Η C C | —O C H C C —OH (1) h2 o m丨 h2 o n (式中，m及η表示1以上之整數）表示之包含3-輕基丁酸及 3-羥基己酸之共聚合聚酯P(3HB-co-3HH)。 【實施方式】 以下，詳細地說明本發明。 本發明之聚酯合成酵素表現質體（I)為包含於 100136.doc 11 200540269
PHB-4/pJRDdTc+149NS171DG(FERM BIM0259 及 FIRDI BCRC 940479)，能以高生產性合成高3HH組成型p(3HB_ co_3HH)之酵素之表現質體。 表現質體之製作 • 首先，說明本發明之聚酯合成酵素表現質體⑴之製作。 全部基因操作可依照Molecular Cloning (ceid SpHng Harbor Laboratory Press，（1989))記載之方式進行。又，基 φ 因操作所使用之酵素及選殖宿主等，可從市場之供應者購 入，依照其之說明而使用。再者，酵素只要可使用於基因 操作者即可，並無特別限定。又，選殖宿主並無特別限定， 可為例如大腸菌DH5a株等。 本發明中，載體可使用廣宿主範圍載體之一之pJRD215 (ATCC 37533)之衍生物 PJRDdTc (記載於 WO04/074476) 等。本表現質體構築所使用之載體之序列，如序列編號4 所示。 • 聚酯合成酵素基因，可調製序列編號3所示之從豚鼠產氣 單胞桿菌（Aeromonas caviae)而來之N149S/D171G二重變異 體基因片段做為限制酵素EcoRI片段，插入載體之相同限制 酵素部位構築而成。再者，N149S變異體基因及di7ig變異 體基因’如 T· Kichise 等 Appl. Environ· Microbiol· 68, 2411-2419 (2002)中所記載，分別將第149號胺基酸之天冬 醯胺用絲胺酸取代，第171號胺基酸之天冬胺酸用甘胺酸取 代之從豚鼠產氣單胞桿菌而來之聚酯合成酵素基因·。圖^ 表示構築本發明之聚酯合成酵素表現質體（I)之順序。 100136.doc 12 200540269 聚酯合成酵素基因，除構造基因外，只要具有啟動子及 終止子等且在宿主菌中有機能之表現單元即可。再者，聚 酯合成酵素表現質體中，上述表現單元可存在1個以上，亦 可存在複數個。 再者，本發明之包含於PHB-4/pJRDdTc+149NS171DG (FERM BP-10259及FIRDI BCRC 940479)之聚酉旨合成酵素表 現質體（I)，於本說明書中以「pJRDdTc+149NS171DG」表 示。 繼而，關於本發明之聚酯合成酵素表現質體（II)之製作加 以說明。 本發明之聚酯合成酵素表現質體（II)為將包含於 PHB-4/pJRDdTc+149NS171DG(FERM BP-10259 及 FIRDI BCRC 940479)之聚酯合成酵素表現質體（I)小型化者。 將質體小型化，係藉由使聚酯合成酵素基因之表現及使 質體複製中不需要之部分失去而進行。例如，若使用之質 體中具有抗生物質耐性基因2個以上，則可刪除其中任何一 個。 在本發明質體之情況，雖可刪除康納黴素（kanamycin)而才 性基因或鏈黴素耐性基因，然而以刪除鏈黴素耐性基因為 較佳。此種使特定基因失去之方法，可使用利用限制酵素 部位之方法或藉由PCR之方法。 又，亦可適當地使用欠缺接合傳達能力之一部分或全 部，且欠缺鏈黴素耐性基因之聚酯合成酵素表現質體。 本發明之聚酯合成酵素表現質體（II)藉由將質體小型 100136.doc -13- 200540269 化’當將表現質體導入宿主時，可使轉形率提高。 轉形體之製作 繼而，有關轉形體之製作加以說明。 本舍明之轉幵> 冑，為藉由上述聚醋合成酵素表現質體⑴ •至（Π)任何一項轉形者。總之，本發明之轉形體，為藉由將 上述聚醋合成酵素表現質體⑴至（Π)任何一項導入適合於 該質體之宿主中而得到者。 • 宿主並無特別限制，可使用從自然界單離之微生物，或 寄存於菌株寄託機關（例如IFO或ATCC等）之微生物等。具 體而a ，可使用如雷爾氏菌（Ralst〇nia)屬、產氣單胞桿菌 (Aeromonas)屬、埃希氏菌（Escherichia)屬產鹼菌 (Alcaligenes)屬或假單胞菌（pseud〇m〇nas)屬等細菌類之非 聚醋生產菌。從安全性及生產性之觀點而言以雷爾氏菌屬 為較佳而以真養雷爾氏菌（Ralstonia eutropha)為更佳，尤 其以藉由變異劑處理或者基於同源重組之基因破壞處理而 • 將聚酯合成酵素失活之真養雷爾氏菌（例如真養雷爾氏菌
PHB_4株等）為特佳。真養雷爾氏菌PHB-4株可從例如DSMZ 荨機關取得。 將聚酯合成酵素表現質體導入微生物，可依照公知之方 法進行。例如，可使用電穿孔法（Current Pr〇t〇c〇is h M〇recular Biology，1卷，h84 頁，1994 年），或鈣法 (Lederberg· Ε·Μ· et al·，J. Baceteriol. 119. 1072 (1974))等。 本發明之較佳轉形體，例如為將聚酯合成酵素表現質體 (I)導入為宿主之真養雷爾氏菌之轉形體等。具體而言，如 100136.doc -14- 200540269 以下所不之轉形體等。為將pJRDdTc+149NS171DG導入真
養雷爾氏菌PHB-4株之轉形體之PHB-4/pJRDdTc + 149NS171DG(寄存編號：FERM BP-10259，寄存日：曰本 平成17年2月23日）。再者，該轉形體係於日本國茨城縣筑 波市東1 丁目1番地丨中央第6之獨立行政法人產業技術綜合 研究所專利生物寄存中心，根據布達佩斯條約進行國際寄 子 本案申睛人另以
PHB-4/pJRDdTc+149NS171DG 之名稱，於2005年 6 月 23 日再 將該轉形體寄存於中華民國（台灣）食品工業發展研究所 (FIRDI)，其寄存編號為bcrc 940479 〇 P(3HB-co-3HH)生產 繼而，說明P(3HB-co-3HH)之生產。 本务明之聚S曰製造方法，為使用上述轉形體者。 上述聚酯以下式（1)
Η Γ ch3 〕 Γ C3H7 Λ 1 1 一 0 一 CH 一 C 一 C 一 -O — CH — c — C 一 h2 〇 m H2 〇 •OH (1) (式中，m及η表示1以上之整數）表示之包含3_羥基丁酸及 3-羥基己酸之共聚合聚酯P(3HB_c〇_3HH)為較佳，而以高 3HH組成之共聚合聚酯p(3HB_c〇_3HH)為更佳。 關於構成高3HH組成P(3HB-co-3HH)之各單体單元之組 成比，並無特別限定，然而從柔軟性良好度而言，以3HH 單元為8莫耳。/。以上且為50莫耳％以下為較佳，而以為1〇莫 耳％以上且為40莫耳。/。以下為更佳，以為12莫耳％以上且為 100136.doc -15- 200540269 30莫耳％以下為特佳。 在生產P(3HB-CO-3HH)時，給與糖、油脂或脂肪酸做為碳 源’並使用含有為碳源以外營養源之氮源，無機鹽類及其 他有機營養源之培養基來培養上述轉形體。 舉例言之’用於培養以屬於雷爾氏菌屬、產氣單胞桿菌 屬、埃希氏菌屬、產鹼菌屬或假單胞菌屬微生物等細菌為 伯主所得到之轉形體之培養基，可使用能提供微生物可利 用之破源，且視情況將氮源、無機鹽類及有機營養源中之 任一者加以限制之培養基，例如使用氮源限制於0.01至 〇·ι%之培養基等。 糖可為例如葡萄糖或果糖等碳水化合物。 油脂多為含有碳數10以上之飽和或不飽和脂肪酸之油 脂’例如椰子油、棕櫚油或棕櫊核油等。 脂肪酸可為己酸、辛酸、癸酸、月桂酸、油酸、棕櫚酸、 亞麻油^、亞麻油稀酸（linolenic acid))或肉莖窺酸等飽和 或不飽和脂肪酸，或者此等脂肪酸之酯或鹽等脂肪酸衍生 物。 氮源除為例如氨、氣化銨、硫酸銨或磷酸銨等銨鹽之外， 可為蛋白胴、肉萃取液或酵母萃取液等。 無機鹽類可為例如磷酸二氫鉀、磷酸氫鉀、麟酸鎂、硫 酸鎂或氣化鈉等。 其他有機營養源，可為例如甘胺酸、丙胺酸、絲胺酸、 禾胺馱或脯胺酸等胺基酸，或維生素B 1、維生素B 12或維 生素C等維生素等。 100136.doc -16- 200540269 又’培養液中亦可添加與本發明表現質體中存在之抗生 物質耐性基因對應之抗生物質（康納黴素）。 • 培養溫度只要為該菌可繁殖之溫度即可，不過以20°C至 4〇°C為較佳。培養時間並無特別限定，不過以1至7日左右 * 為較佳。 然後，從所得到之該培養菌體回收p(3hb_c〇_3hh)即可。 本發明中，從菌體回收p(3HB_c〇_3HH)可依照例如以下之 Φ 方法而進行。培養終了後，藉由離心器等從培養液分離菌 體，將该菌體藉由蒸餾水或甲醇等洗淨，並使其乾燥。使 用氯仿等有機溶劑從該乾燥菌體萃取P(3HB-CO-3HH)。從含 有該P(3HB-C0-3HH)之有機溶劑溶液，藉由過濾等除去菌體 成刀’並於該濾液中添加甲醇或己烷等弱溶媒，使 P(3HB-CO-3HH)沉澱。再者，藉由過濾及離心除去上清液， 並使其乾燥及回收p(3HB-co-3HH)。 得到之P(3HB-CO-3HH)之重量平均分子量（Mw)及3HH組 籲成（莫耳％)之分析，可藉由例如氣體層析法或核磁共振法等 而進行。或者，可利用使用尼羅紅（Nilered)之染色法確認 聚酉曰生產之簡易法。亦即，在重組菌繁殖之瓊脂培養基中 添加尼羅紅，將重組菌培養丨至7日，藉由觀察重組菌是否 變紅’可確認有無聚酯之生產。 [發明之效果] 如上所述，藉由使用新穎聚酯合成酵素表現質體產生之 真養雷爾氏菌之轉形體，即使在廉價之植物油脂等單一碳 源下培養，亦可安全且高生產性地生產適合加工成薄膜狀 100136.doc -17- 200540269 之高 3HH組成之P(3HB-co-3HH)。 [為實施發明之最佳形態] 以下，藉由實施例詳細地說明本發明。然而，本發明並 不將其技術範圍限定於此等實施例。 (實施例1)表現質體之構築 高3HH組成P(3HB-co-3HH)生產用之表現質體如下述構 pJRDdTc+149NS171DG構築用之載體，可使用藉由序列 編號4所示之DNA編碼之載體。本載體可與w〇〇4/〇74476記 載之pJRDdTc同樣地構築。 為聚酯合成酵素基因之從豚鼠產氣單胞桿菌而來之 Nl49S/DmG二重變異體基因片段，可藉由pcR法製成。 N149S變異及D171G變異係分別將從豚鼠產氣單胞桿菌而 來之PHA合成酵素之第149號胺基酸之天冬醯胺用絲胺酸 取代，第1 71號胺基酸之天冬胺酸用甘胺酸取代。因此，使 用非專利文獻10中記載之將從豚鼠產氣單胞桿菌而來之 N149S變異基因分段選殖於PUC19之EcoRI部位，如序列編 號1及序列編號2所示之合成DNA做為引子，進行PCR。其
條件為（1)94°C2 分鐘，（2)94°C30 秒，（3)55艺30 秒，（4)72°C 2分鐘，從（2)至（4)進行25循環，（5)72°C5分鐘；聚合酶係使 用LA Taq聚合酶（寶生公司製）。用限制酵素EcoRI切斷，調 製序列編號3所示之N149S/D171G片段，將本載體插入用相 同酵素切斷之部位，構築成表現質體pjRDdTc+ 149NS171DG。 100136.doc -18- 200540269 (實施例2)轉形體之製作 藉由電脈衝法製作實施例1得到之含有表現質體之真養 雷爾氏菌PHB-4株轉形體。總之，基因導入裝置係使用 Biorad公司製之基因解譯器（Geneparser)，比色管（cuvet)同 樣地使用Biorad公司製之間隙（gap) 〇·2 cm者。於比色管中 注入勝任細胞（competent cell)4〇〇 μ1及表現質體2〇 μ卜安裝 於脈衝裝置’以靜電容量25 pF，電壓1.5 kV，電阻值8 〇 〇 Ω之條件進行電脈衝。脈衝後，將比色管中之菌液於
NutrientBroth培養基（DIFCO公司製）中，在30°C下震盪培養 3小時’再於選擇平板（NutrientAgar培養基（DIFC〇公司 製），康納黴素100 mg/L)上，於3〇°C培養2日，取得轉形體。 (實施例3)轉形體之選擇 將實施例2所得到之轉形株接種於含有尼羅紅之培養基 (磷酸氫二鈉· 12水合物9g、磷酸二氫鉀L5 g、氣化銨〇〇5 g、硫酸鎂· 7水合物0.02g、果糖〇·5 g、氣化鈷· 6水合物 〇·25 ppm、氣化鐵（in) · 6水合物16 、氣化弼· 2水合物 10 ·3 ppm、氣化鎳· 6水合物0.12 ppm、硫酸銅· 5水合物〇.16 ppm、尼羅紅〇·5 mg及瓊脂15 g/1 L)，並於3〇(>c培養一週。 結果，從菌落變紅確認菌體内積蓄聚酯，選擇該菌落，進 行 P(3HB-CO-3HH)之生產。該轉形體以 PHB_4/pJRDdTc+ 149NS171DG (寄存編號：FERM BP-10259，寄存日：平成 17年2月23曰）之名稱，寄存於日本國茨城縣筑波市東1丁目 1番地1中央第6之獨立行政法人產業技術綜合研究所專利 生物寄存中心，根據布達佩斯條約進行國際寄託。又，本 100136.doc -19- 200540269 案申請人另以 jRak/oe/a PHB-4/pJRDdTc+ 149NS1 7 IDG之名稱，於2005年6月23日再將該轉形體寄存 於中華民國（台灣）食品工業發展研究所（FIRDI)，其寄存編 號為 BCRC 940479。 (實施例4) P(3HB-co-3HH)之生產及精製
種母培養基之組成為1 w/v%肉萃取液、1 w/v°/q細菌培養 用胰蛋白胴（Bacto-Trypton)、0.2 w/v%酵母萃取液、0.9 w/v% Na2P04 · 12H20 及 0.15 w/v% KH2P〇4，並調為 pH 6.8。 前培養基之組成為 1.1 w/v% Na2P04 · 12H20、0.19 w/v°/〇 ΚΗ2Ρ〇4、1·29 w/v% (NH4)2S04、0·1 w/v% MgS04 · 7Η20、 2.5 w/v%含油酸棕橺油、0.5 v/v微量金屬鹽溶液（在0.1N鹽 酸中將 1.6 w/v% FeCl3 · 6Η20、1 w/v% CaCl2 · 2Η20、0.02 w/v% CoCl2· 6H20、0.016 w/v %CuS04· 5H20及 0.012 w/v% NiCl2 · 6H20溶解者）及5xl0_6w/v%康納黴素。 P(3HB-co-3HH)生產培養基之組成為0.385 w/v% Na2P04 · 12H20、0.067 w/v% KH2P〇4、0-291 w/v% (NH4)2S04、0.1 w/v% MgS04 · 7H20、0.5 v/v%微量金屬鹽 溶液（在 0.1N鹽酸中溶解有 1·6 w/v% FeCl3 · 6H2〇、1 w/v°/〇 CaCl2· 2H20、0.02 w/v% CoCl2· 6H20、0.016 w/v% CuS04 · 51120及0.012胃/¥%犯(：12-6}120者）、0.〇5\¥/¥%3108?1；11丑\ 200K(消泡劑，Cognis日本公司製）及5xl(T6w/v%康納黴素。 碳源係使用為將棕櫚核油分離低熔點部分之棕櫚核油酸油 做為唯一碳源，在培養全程，以比基質供應速度為0.08至 〇.1(油脂（g))x(乾燥菌體淨重（g))-lx(小時（h))-1之方式進行 100136.doc •20- 200540269 流力口。 將PHB-4/pJRDdTc+149NS171DG轉形體之甘油貯液 (glycerol stock)(5 0 μΐ)接種於種母培養基（10 ml)，並培養24 小時，再以1.0 v/v接種於内裝1.8 L前培養培養基之3 L罐式 發酵槽（jar fermentor)(丸菱生工（Marubishi Bioengi)公司 製，MDL-300型）中。運轉條件為培養溫度30°C，攪拌速度 500 rpm，通氣量1.8 L/min，pH控制在6.7與6.8之間，同時 培養28小時。pH控制組係使用7%氳氧化銨水溶液。 P(3HB-co_3HH)生產培養，係將前培養種母以5.0 v/v%% 接種於内裝6L生產培養基之10L罐式發酵槽（丸菱生工公司 製，MDL-1000型）中。運轉條件為培養溫度28°C，攪拌速 度400 rpm，通氣量3.6 L/min，pH控制在6.7與6.8之間。pH 控制係使用7%氫氧化銨水溶液。培養進行約60小時，培養 終了後，藉由離心回收菌體，用甲醇洗淨後，冷凍乾燥， 測定乾燥菌體重量。 在得到之乾燥菌體1 g中添加1 〇〇 ml氯仿，並於室溫攪拌 一晝夜，萃取菌體内之P(3HB-co-3HH)。過濾菌體殘餘物並 用蒸發器（evaporator)濃縮至總容積成為30 ml後，慢慢添加 約90 ml之己烷，並緩慢攪拌，同時放置1小時。過濾析出 之P(3HB-co-3HH)後，在50°C真空乾燥3小時。測定乾燥 P(3HB-co-3HH)之重量，並算出菌體内含量。結果 PHB-4/pJRDdTc+149NS171DG轉形體之P(3HB-co-3HH)含 量在62小時為66.1(重量％)之「高含量」。 (實施例5) P(3HB-co-3HH)之3HH組成分析 100136.doc -21 - 200540269 藉由轉形體 PHB-4/pJRDdTc+149NS171DG 生產之 P(3HB-co-3HH)之3HH組成之分析如下述使用氣體層析法測定。 於約20 mg之乾燥P(3HB-co-3HH)中添加2 ml之硫酸-甲 醇混合液（15 : 85)及2 ml之氯仿並密封，在100°C加熱140 分鐘，得到P(3HB-co-3HH)分解物之甲S旨。冷卻後，於其中 逐次少量添加1.5 g之碳酸氫納進行中和，放置直到停止產 生二氧化破。添加4 ml之二異丙醚並充分混合後，離心， 藉由毛細管氣體層析法分析上清液中P(3HB-co-3HH)分解 物之單體單元組成。氣體層析係使用島津製作所之GC-17， 毛細管管柱係使用GL Science公司製之NEUTRA BOND -1(管柱長25 m，管柱内徑0.25 mm，液膜厚0.4 μπι)。 使用He做為遞送氣體（carrier gas)，管柱入口壓力調為 100 kPa，將樣本1 μΐ注入。溫度條件為從起始溫度100°C至 200°C為止以8°C /分之速度升溫，再者200°C至290°C為止以 30°C/分之速度升溫。用上述條件分析之結果，62小時培養 終了時之P(3HB-co-3HH)之3HH組成，為14.7(莫耳％)，係 屬適合加工成薄膜之高3HH組成。 如上所述，藉由使用新穎聚酯合成酵素表現質體產生之 真養雷爾氏菌轉形體，即使在廉價之植物油脂等單一碳源 下培養，亦可安全且高生產性地生產適於加工成薄膜狀等 之高 3HH組成型 P(3HB-co_3HH)。 【圖式簡單說明】 圖 1 為本發明之pJRDdTc+149NS171DG (FERM BP-10259 及FIRDI BCRC 940479)聚酯合成酵素表現質體之構築圖。 100136.doc -22- 200540269 序列表 <11 <12' 分有限公司(KANEKA CORPORATION) <130>B040191TW01 <140)094111212 <141)2005-04-08 <150>JP2004-115642 <151)2004-4-9 <160>4 <210>1 <211>26 <212>DNA <213〉人造序列
<220> <223〉引子 <400>1 accctggagt ccggcggcca gaacct 26 <210>2 <211>26 <212>DNA <213〉人造序列 <220〉 <223〉引子 <400>2 aggttctggc cgccggactc cagggt 26
<210〉 3 <211〉 594 <212〉 PRT <213>豚鼠產氣單胞桿菌 <220〉 <223〉聚羥基烷酸酯合成酵素之變異株 <400〉 9
Met Ser Gin Pro Ser Tyr Gly Pro Leu Phe Glu Ala Leu Ala His Tyr 15 10 15
Asn Asp Lys Leu Leu Ala Met Ala Lys Ala Gin Thr Glu Arg Thr Ala 20 25 30 6ln Ala Leu Leu Gin Thr Asn Leu Asp Asp Leu Gly Gin Val Leu Glu 100136.doc 200540269 35 40 45
Gin Gly Ser Gin Gin Pro Trp Gin Leu lie Gin Ala Gin Met Asn Trp 50 55 60
Trp Gin Asp 6ln Leu Lys Leu Met Gin His Thr Leu Leu Lys Ser Ala 65 70 75 80
Gly Gin Pro Ser Glu Pro Val lie Thr Pro Glu Arg Ser Asp Arg Arg 85 90 95
Phe Lys Ala Glu Ala Trp Ser Glu Gin Pro lie Tyr Asp Tyr Leu Lys 100 105 110
Gin Ser Tyr Leu Leu Thr Ala Arg His Leu Leu Ala Ser Val Asp Ala 115 120 125
Leu Glu Gly Val Pro Gin Lys Ser Arg Glu Arg Leu Arg Phe Phe Thr 130 135 140
Arg Gin Tyr Val Ser Ala Met Ala Pro Ser Asn Phe Leu Ala Thr Asn 145 150 155 160
Pro Glu Leu Leu Lys Leu Thr Leu Glu Ser Gly Gly Gin Asn Leu Val 165 170 175
Arg Gly Leu Ala Leu Leu Ala Glu Asp Leu Glu Arg Ser Ala Asp Gin 180 185 190
Leu Asn lie Arg Leu Thr Asp Glu Ser Ala Phe Glu Leu Gly Arg Asp 195 200 205
Leu Ala Leu Thr Pro Gly Arg Val Val Gin Arg Thr Glu Leu Tyr Glu 210 215 220
Leu lie Gin Tyr Ser Pro Thr Thr Glu Thr Val Gly Lys Thr Pro Val 225 230 235 240
Leu lie Val Pro Pro Phe lie Asn Lys Tyr Tyr lie Met Asp Met Arg 245 250 255
Pro 6ln Asn Ser Leu Val Ala Trp Leu Val Ala Gin Gly Gin Thr Val 100136.doc 200540269 260 265 270
Phe Met lie Ser Trp Arg Asn Pro Gly Val Ala Gin Ala Gin lie Asp 275 280 285
Leu Asp Asp Tyr Val Val Asp Gly Val lie Ala Ala Leu Asp Gly Val 290 295 300 6lu Ala Ala Thr Gly 6lu Arg Glu Val His Gly lie Gly Tyr Cys lie 305 310 315 320
Gly Gly Thr Ala Leu Ser Leu Ala Met Gly Trp Leu Ala Ala Arg Arg 325 330 335
Gin Lys Gin Arg Val Arg Thr Ala Thr Leu Phe Thr Thr Leu Leu Asp 340 345 350
Phe Ser Gin Pro Gly Glu Leu Gly lie Phe lie His Glu Pro lie lie 355 360 365
Ala Ala Leu Glu Ala Gin Asn Glu Ala Lys Gly lie Met Asp Gly Arg 370 375 380
Gin Leu Ala Val Ser Phe Ser Leu Leu Arg Glu Asn Ser Leu Tyr Trp 385 390 395 400
Asn Tyr Tyr lie Asp Ser Tyr Leu Lys Gly Gin Ser Pro Val Ala Phe 405 410 415
Asp Leu Leu His Trp Asn Ser Asp Ser Thr Asn Val Ala Gly Lys Thr 420 425 430
His Asn Ser Leu Leu Arg Arg Leu Tyr Leu Glu Asn Gin Leu Val Lys 435 440 445
Gly Glu Leu Lys lie Arg Asn Thr Arg lie Asp Leu Gly Lys Val Lys 450 455 460
Thr Pro Val Leu Leu Val Ser Ala Val Asp Asp His lie Ala Leu Trp 465 470 475 480
Gin Gly Thr Trp Gin Gly Met Lys Leu Phe Gly Gly Glu Gin Arg Phe 100136.doc 200540269 485 490 495
Leu Leu Ala Glu Ser 6ly His lie Ala Gly lie lie Asn Pro Pro Ala 500 505 510
Ala Asn Lys Tyr Gly Phe Trp His Asn Gly Ala Glu Ala Glu Ser Pro 515 520 525
Glu Ser Trp Leu Ala Gly Ala Thr His Gin Gly Gly S,er Trp Trp Pro 530 535 540
Glu Met Met Gly Phe lie Gin Asn Arg Asp Glu Gly Ser Glu Pro Val 545 550 555 560
Pro Ala Arg Val Pro Glu Glu Gly Leu Ala Pro Ala Pro Gly His Tyr 565 570 575
Val Lys Val Arg Leu Asn Pro Val Phe Ala Cys Pro Thr Glu Glu Asp 580 585 590
Ala Ala <210>4 <211>9141
<212>DNA <213>pJRDdTc <220〉
<223〉PJRDdTc之變異株 <400>4 60 120 180 240 300 360 420 480 aactgcacat tcgggatatt tctctatatt cgcgcttcat cagaaaactg aaggaacctc cattgaatcg aactaatatt ttttttggtg aatcgcattc tgactggttg cctgtcagag gcggagaatc tggtgatttt gtttttcgac gtggtgacgg gcatgccttc gcgaaaatcg cacctgcttc ccgccgcggt gagctcgctg gagagcgtga ccgcctcatt tggctcaaag gtcgaggtgt ggcttgcccc gaggtcatca actggcagga ggaacaggag ggtgcatgct tggtgataac ggcaattccg ggagtaccgg cggctgatct gtctggagcg gatttgctca aagcgtggcc gtcaatgggg cagcaacttg gcgctgttca cagcctatcg gttgatcaat gtccgtttga gcgcaggctg tcgcgaatgt tcggacgcgc cgttgatgtg gtgtcccgca atgccgtcaa tcccgacttc ttaccggacg aggacaagag tacgccgcag ctcgatcttt 100136.doc -4- 540 600 600
200540269 tggctcgtgt cgaacgagag ctaccggtgc ggctcgacca agagcgcacc gatatggttg tttgccatgg tgatccctgc atgccgaact tcatggtgga ccctaaaact cttcaatgca cgggtctgat cgaccttggg cggctcggaa cagcagatcg ctatgccgat ttggcactca tgattgctaa cgccgaagag aactgggcag cgccagatga agcagagcgc gccttcgctg tcctattcaa tgtattgggg atcgaagccc ccgaccgcga acgccttgcc ttctatctgc gattggaccc tctgacttgg ggttgatgtt catgccgcct gtttttcctg ctcattggca cgtttcgcaa cctgttctca ttgcggacac cttttccagc ctcgtttgga aagtttcatt gccagacggg actcctgcaa tcgtcaaggg attgaaacct atagaagaca ttgctgatga actgcgcggg gccgactatc tggtatggcg caatgggagg ggagcagtcc· ggttgctcgg tcgtgagaac aatctgatgt tgctcgaata tgccggggag cgaatgctct ctcacatcgt tgccgagcac ggcgactacc aggcgaccga aattgcagcg gaactaatgg cgaagctgta tgccgcatct gaggaacccc tgccttctgc ccttctcccg atccgggatc gctttgcagc tttgtttcag cgggcgcgcg atgatcaaaa cgcaggttgt caaactgact acgtccacgc ggcgattata gccgatcaaa tgatgagcaa tgcctcggaa ctgcgtgggc tacatggcga tctgcatcat gaaaacatca tgttctccag tcgcggctgg ctggtgatag atcccgtcgg tctggtcggt gaagtgggct ttggcgccgc caatatgttc tacgatccgg ctgacagaga cgacctttgt ctcgatccta gacgcattgc acagatggcg gacgcattct ctcgtgdgct ggacgtcgat ccgcgtcgcc tgctcgacca ggcgtacgct tatgggtgcc tttccgcagc ttggaacgcg gatggagaag aggagcaacg cgatctagct atcgcggccg cgatcaagca ggtgcgacag acgtcatact agatatcaag cgacttctcc tatcccctgg gaacacatca atctcaccgg agaatatcgc tggccaaagc cttagcgtag gattccgccc cttcccgcaa acgaccccaa acaggaaacg cagctgaaac gggaagctca acacccactg acgcatgggt tgttcaggca gtacttcatc aaccagcaag gcggcacttt cggccatccg ccgcgcccca cagctcgggc agaaaccgcg acgcttacag ctgaaagcga ccaggtgctc ggcgtggcaa gactcgcagc gaacccgtag aaagccatgc tccagccgcc cgcattggag aaattcttca aattcccgtt gcacatagcc cggcaattcc tttccctgct ctgccataag cgcagcgaat gccgggtaat actcgtcaac gatctgatag agaagggttt gctcgggtcg gtggctctgg taacgaccag tatcccgatc ccggctggcc gtcctggccg ccacatgagg catgttccgc 100136.doc 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220
200540269 gtccttgcaa tactgtgttt acatacagtc tatcgcttag cggaaagttc ttttaccctc agccgaaatg cctgccgttg ctagacattg ccagccagtg cccgtcactc ccgtactaac tgtcacgaac ccctgcaata actgtcacgc ccccctgcaa taactgtcac gaacccctgc aataactgtc acgcccccaa acctgcaaac ccagcagggg cgggggctgg cggggtgttg gaaaaatcca tccatgatta tctaagaata atccactagg cgcggttatc agcgcccttg tggggcgctg ctgcccttgc ccaatatgcc cggccagagg ccggatagct ggtctattcg ctgcgctagg ctacacaccg ccccaccgct gcgcggcagg gggaaaggcg ggcaaagccc gctaaacccc acaccaaacc ccgcagaaat acgctggagc gcttttagcc gctttagcgg cctttccccc tacccgaagg gtgggggcgc gtgtgcagcc ccgcagggcc tgtctcggtc gatcattcag cccggctcat ccttctggcg tggcggcaga ccgaacaagg cgcggtcgtg gtcgcgttca aggtacgcat ccattgccgc catgagccga tcctccggcc actcgctgct gttcaccttg gccaaaatca tggcccccac cagcaccttg cgccttgttt cgttcttgcg ctcttgctgc tgttcccttg cccgcacccg ctgaatttcg gcattgattc gcgctcgttg ttcttcgagc ttggccagcc gatccgccgc cttgttgctc cccttaacca tcttgacacc ccattgttaa tgtgctgtct cgtaggctat caaggaggca cagcggcggc aatcccgacc ctactttgta gcctaacggc cacccttcgg gcggtgcgct ctccgagggc cattgcatgg agccgaaaag caaaagcaac agcgaggcag catggcgatt tatcacctta cggcgaaaac cggcagcagg tcgggcggcc aatcggccag ggccaaggcc gactacatcc agcgcgaagg caagtatgcc cgcgacatgg atgaagtctt gcacgccgaa tccgggcaca tgccggagtt cgtcgagcgg cccgccgact actgggatgc tgccgacctg tatgaacgcg ccaatgggcg gctgttcaag gaggtcgaat ttgccctgcc ggtcgagctg accctcgacc agcagaaggc gctggcgtcc gagttcgccc agcacctgac cggtgccgag cgcctgccgt atacgctggc catccatgcc ggtggcggcg agaacccgca ctgccacctg atgatctccg agcggatcaa tgacggcatc gagcggcccg ccgctcagtg gttcaagcgg tacaacggca agaccccgga gaagggcggg gcacagaaga ccgaagcgct caagcccaag gcatggcttg agcagacccg cgaggcatgg gccgaccatg ccaaccgggc attagagcgg gctggccacg acgcccgcat tgaccacaga acacttgagg cgcagggcat cgagcgcctg cccggtgttc acctggggcc gaacgtggtg gagatggaag gccggggcat ccgcaccgac cgggcagacg tggccctgaa 100136.doc 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 200540269
catcgacacc tgaacgcaat aaccgctggc cccagcaggc cggcatgggt cgcagagcgc tggcttccat cgccactgac caggactttg cggcgtcagg gctccagaac gcccgccgag ggatgacatg ctatcaggca ccggacgctg ccgcttggcg gtgggtgctg gcaggctggc cagcctcgaa ccgcagcgag tatcgccgcg ggccgaggcc gcgcaccgtc acgggcaccg gtgcttgcgt tatggaatac ttggttgccc cgctgatatg gccaacgccc cgacagagtg ccagagcatg cagcgaggcg ggagctggcc ccggagcgcg gatgcctacg aaccgaggac caggccatag gacgccacca acgccatggc caggagcggc aaagccgagg tgggtcaagg gccagcgagt ggcttcacca ctgcgtgaat cagcagatcg ctgcccgagc atggccgggc cagaagctgg agcccagcgc agcaaggtca gcaccccgcc tggtactcac gaaaaaagcg ggtggctttg gccagcctta agatcatcga aagaaatcca gcgacactgg caggcggcgg aaagagttgc tcgcgggggt gtggtgctgc ggctcgatct gccgacagct ccggccagat tcaagcggat atggtctggt gccgggagcc tggccgacgc acgacgccga accgcaagga ccaagggcaa agcaggccca ggcagcttag tggtcaagcg ccagccgggg tggcagagcg tgggtctgcc agcgaggcat gcctgttata cttcagggtc gttatacgtc accttgacgg cttacaggaa gaggcatcaa ccgacgaagc agtggctgaa gggaggtagc ggcactggaa tgatcgcatc ggctgcgctt caaggccatg gatgaaccgg gaatgcccag gctggtggac tgccctggta cgcaggcggt cccggccagc caagcacacc gaccgccacc gcggcagcag ccgccaccgg cttcggtgat ccgcagtgcc caagcccggc cagcgtccag ggacaggggc ctatgagtac ggtctacctg aaacaaggcc ctgcaccttg taccgggagg cgagttagcg ccggcaggtc agcccagcgc cgccgaggtg gctatggcta gttgctcttg ggtggcccga ggctgtgagc gaatggtcag ggcaatgacg gacctcagcg gtggaaacca gaacttcggg gccgacagcc acccgcgccg gctggcccgg gagaaggccc cgcacggcgc gabctcagca gaggaaatcg cacgaagcgg cttgcgcggg gggccagatt tcacgcacag atcaaaagtg gaggctggcc tccttgttcc caatagacca gagcagatcg atgagccaga cagacagggg agcagcgccg accgtgatgc ctcgacctga tgaagaacga gcttcgatat ccgccgaagt tgtatatcag agtttgacct gcccgaagaa ggcagattgc gccactatgg gttatcagcc cgctggtgca gcaggctggc tggacgagta agtgcgactt gcaaggccat attacatcga ccgagctggc tcagcatgta aagggggttt acaagggcta gcttttcagt gcgaagacaa 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 100136.doc
200540269 gcctttcggc cccggcaagt ttctcggtga ctgatatgaa agaccaaaag gacaagcaga ccggcgacct gctggccagc cctgacgctg tacgccaagc gcgatatgcc gagcgcatga aggccaaagg gatgcgtcag cgcaagttct ggctgaccga cgacgaatac gaggcgctgc gcgagtgcct ggaagaactc agagcggcgc agggcggggg tagtgacccc gccagcgcct aaccaccaac tgcctgcaaa ggaggcaatc aatggctacc cataagccta tcaatattct ggaggcgttc gcagcagcgc cgccaccgct ggactacgtt ttgcccaaca tggtggccgg tacggtcggg gcgctggtgt cgcccggtgg tgccggtaaa tccatgctgg ccctgcaact ggccgcacag attgcaggcg ggccggatct gctggaggtg ggcgaactgc ccaccggccc ggtgatctac ctgcccgccg aagacccgcc caccgccatt catcaccgcc tgcacgccct tggggcgcac ctcagcgccg aggaacggca agccgtggct gacggcctgc tgatccagcc gctgatcggc agcctgccca acatcatggc cccggagtgg ttcgacggcc tcaagcgcgc cgccgagggc cgccgcctga tggtgctgga cacgctgcgc cggttccaca tcgaggaaga aaacgccagc ggccccatgg cccaggtcat cggtcgcatg gaggccatcg ccgccgatac cgggtgctct atcgtgttcc tgcaccatgc cagcaagggc gcggccatga tgggcgcagg cgaccagcag caggccagcc ggggcagctc ggtactggtc gataacatcc gctggcagtc ctacctgtcg agcatgacca gcgccgaggc cgaggaatgg ggtgtggacg acgaccagcg ccggttcttc gtccgcttcg gtgtgagcaa ggccaactat ggcgcaccgt tcgctgatcg gtggttcagg cggcatgacg gcggggtgct caagcccgcc gtgctggaga ggcagcgcaa gagcaagggg gtgccccgtg gtgaagccta agaacaagca cagcctcagc cacgtccggc acgacccggc gcactgtctg gcccccggcc tgttccgtgc cctcaagcgg ggcgagcgca agcgcagcaa gctggacgtg acgtatgact acggcgacgg caagcggatc gagttcagcg gcccggagcc gctgggcgct gatgatctgc gcatcctgca agggctggtg gccatggctg ggcctaatgg cctagtgctt ggcccggaac ccaagaccga aggcggacgg cagctccggc tgttcctgga acccaagtgg gaggccgtca ccgctgatgc catggtggtc aaaggtagct atcgggcgct ggcaaaggaa atcggggcag aggtcgatag tggtggggcg ctcaagcaca tacaggactg catcgagcgc ctttggaagg tatccatcat cgcccagaat ggccgcaagc ggcaggggtt tcggctgctg tcggagtacg ccagcgacga ggcggacggg cgcctgtacg tggccctgaa ccccttgatc gcgcaggccg tcatgggtgg cggccagcat gtgcgcatca 100136.doc 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260
200540269 gcatggacga ggtgcgggcg ctggacagcg aaaccgcccg cctgctgcac cagcggctgt gtggctggat cgaccccggc aaaaccggca aggcttccat agataccttg tgcggctatg tctggccgtc agaggccagt ggttcgacca tgcgcaagcg ccgccagcgg gtgcgcgagg cgttgccgga gctggtcgcg ctgggctgga cggtaaccga gttcgcggcg ggcaagtacg acatcacccg gcccaaggcg gcaggctgac cccccccact ctattgtaaa caagacattt ttatctttta tattcaatgg cttattttcc tgctaattgg taataccatg aaaaatacca tgctcagaaa aggcttaaca atattttgaa aaattgccta ctgagcgctg ccgcacagct ccataggccg ctttcctggc tttgcttcca gatgtatgct cttctgctcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat gccgacggat ttgcactgcc ggtagaactc cgcgaggtcg tccagcctca ggcagcagct gaaccaactc gcgaggggat cgagcccggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgcctcga ggcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc ctagtatagt ctatagtccg tggaattatt atatttatct ccgacgatat tctcatcagt 100136.doc 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 200540269 gaaatccagg ttatcacagt atcgtcatcc ctgccgggcc ggaattctca tgtttgacag taaattgcta acgcagtcag tcggcaccgt caccctggat tcttgcggga t cttatcatcg ataagcttta gcaccgtgta tgaaatctaa gctgtaggca taggcttggt atgcggtagt 9000 caatgcgctc 9060 tatgccggta 9120 9141
100136.doc 10-
Claims (1)
- 200540269 十、申請專利範圍： 1. 一種聚S旨合成酵素表現質體，其包含於pHB_4/pjRDdTc+ 149NS171DG (FERM BP-10259及 FIRDI BCRC 940479)。 2· 一種聚酯合成酵素表現質體，其係將請求項丨之質體小型 化0 3 · 一種轉形體’其係藉由請求項1或2之質體轉形。 4. 如明求項3之轉形體，其中宿主為非聚酯生產性之雷爾氏 菌屬。 5. 如請求項3或4之轉形體，其中宿主為非聚酯生產性之真 養雷爾氏菌。 6. 如請求項3至5中任一項之轉形體，其中宿主為真養雷爾 氏菌PHB-4株。 7· -種轉形體，其為PHB-4/PJRDdTC+149NS17lDG (PERM BP-10259及 FIRDI BCRC 940479)。 8· 一種聚酯之製造方法，其係使用請求項3至7中任一項之 轉形體。 ~ 9·如請求項8之聚酯之製造方法，其中聚酯為下式 Η 产 ch3 Γ c3h7 ^ 1 1 —〇 —CH — C - • C 一 -Ο - CH —C 一 c 一 L H2 〇 m L · h2 o •OH (式中，m及η表示1以上之整數）表示之包令 、匕3扣羥基丁酸 及3-羥基己酸之共聚合聚酯。 100136.doc
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| JP5650368B2 (ja) * | 2005-10-27 | 2015-01-07 | 株式会社カネカ | 新規プラスミドベクター及びプラスミドを安定に保持する形質転換体 |
| US9051589B2 (en) | 2005-10-27 | 2015-06-09 | Kaneka Corporation | Plasmid vector and transformant stably retaining plasmid |
| JP2007259708A (ja) * | 2006-03-27 | 2007-10-11 | Kaneka Corp | 新規生分解性ポリエステルの製造方法 |
| CN104845927A (zh) * | 2006-07-21 | 2015-08-19 | 株式会社钟化 | 基因取代微生物及使用该微生物的聚酯制造方法 |
| CN101501182A (zh) * | 2006-07-21 | 2009-08-05 | 株式会社钟化 | 基因取代微生物及使用该微生物的聚酯制造方法 |
| JP4960033B2 (ja) * | 2006-07-26 | 2012-06-27 | 株式会社カネカ | 酵素活性を低下させた微生物を用いる共重合ポリエステルの製造方法 |
| US7384766B2 (en) | 2006-07-26 | 2008-06-10 | Kaneka Corporation | Gene-substituted microorganisms, and production method of polyesters using the same |
| EP2669365B1 (en) | 2011-01-27 | 2017-04-26 | Kaneka Corporation | Method for producing high-molecular-weight pha |
| CN114072448A (zh) | 2019-04-26 | 2022-02-18 | 株式会社未来科学 | 聚羟基烷酸及其制备方法 |
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