TW201619374A - 具有產生ｌ－精胺酸的棒狀桿菌屬微生物及使用該微生物產生ｌ－精胺酸的方法 - Google Patents
具有產生ｌ－精胺酸的棒狀桿菌屬微生物及使用該微生物產生ｌ－精胺酸的方法 Download PDFInfo
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- TW201619374A TW201619374A TW104133479A TW104133479A TW201619374A TW 201619374 A TW201619374 A TW 201619374A TW 104133479 A TW104133479 A TW 104133479A TW 104133479 A TW104133479 A TW 104133479A TW 201619374 A TW201619374 A TW 201619374A
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- arginine
- microorganism
- argf
- promoter
- pargc
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Abstract
本發明係有關一種具有產生L-精胺酸能力的棒狀桿菌(Corynebacterium)屬微生物及使用該微生物產生L-精胺酸的方法。
Description
本發明係有關一種具有產生L-精胺酸能力之棒狀桿菌(Corynebacterium)屬微生物及使用該微生物產生L-精胺酸之方法。
L-精胺酸為一種常用於胺基酸補充品、藥品、食品等等之胺基酸，並且相關工業上已要求發展有效之L-精胺酸產生法。
採用傳統生物發酵法產生L-精胺酸之方法為一種直接由碳源與氮源產生L-精胺酸之方法，並已有報告提出各種不同方法，包括使用來自短桿菌(Brevibacterium)或棒狀桿菌(Corynebacterium)屬微生物之經誘導修飾菌株之方法、使用經過細胞融合所發展出加強胺基酸產生能力之細菌細胞株之方法等等。近來，已有報告提出一種使用基因重組菌株之方法，其中使抑制精胺酸生物合成之操縱
子argR表現之基因失活(美國專利案案號7,160,705)，及一種利用argF於精胺酸操縱子中過度表現之方法(韓國專利案案號10-0854234)等等。特定言之，刪除調控精胺酸操縱子之argR之作法已被視為精胺酸產生之重要因素。
依據目前已知之事實，在棒狀桿菌微生物中，涉及精胺酸生物合成之argCJBDFR基因係構成操縱子之型式，會受到細胞內精胺酸之反饋抑制(Vehary Sakanyan等人之Microbiology,142：9-108,1996)，因此會限制其高量L-精胺酸產生。
因此，本發明者在致力於提高L-精胺酸產量時，發現可藉由增強精胺酸操縱子與鳥胺酸胺甲醯基轉化酶之活性，無需任何刪除精胺酸抑制因子(argR)(其係一般已知之重要因子)，而相較於親代L-精胺酸產生菌株可以較高產量產生L-精胺酸，因而完成本發明。
本發明之目的為提供一種屬於棒狀桿菌屬之微生物，其具有增強活性之精胺酸操縱子與鳥胺酸胺甲醯基轉化酶而產生L-精胺酸之能力。
本發明另一個目的為提供一種使用棒狀桿菌屬微生物產生L-精胺酸之方法。
為了達成上述目的，本發明提供一種具有增強活性之精胺酸操縱子與鳥胺酸胺甲醯基轉化酶之能夠
產生L-精胺酸之棒狀桿菌屬之微生物。
本發明中，精胺酸操縱子為由涉及L-精胺酸生物合成機制之酵素所組成之操縱子，特定言之，精胺酸操縱子係由構成L-精胺酸生物合成循環步驟之酵素所組成。明確言之，精胺酸操縱子係由N-乙醯基麩胺醯基磷酸酯還原酶(ArgC)、麩胺酸酯N-乙醯基轉化酶(ArgJ)、N-乙醯基麩胺酸酯激酶(ArgB)、乙醯基鳥胺酸胺基轉化酶(ArgD)、鳥胺酸胺甲醯基轉化酶(ArgF)、與精胺酸抑制因子(ArgR)組成，且此等酵素涉及L-精胺酸生物合成之連續酵素反應。
此等構成精胺酸操縱子之酵素涉及使用L-麩胺酸酯作為前體之最終L-精胺酸生物合成。該麩胺酸酯N-乙醯基轉化酶(ArgJ)使用L-麩胺酸酯作為前體來合成N-乙醯基麩胺酸酯，且其可由argJ基因編碼。特定言之，由N-乙醯基鳥胺酸分解形成L-鳥胺酸而得到乙醯基。已知麩胺酸酯N-乙醯基轉化酶涉及屬於棒狀桿菌屬之微生物中L-精胺酸生物合成之再循環反應。
所產生之N-乙醯基麩胺酸酯藉由N-乙醯基麩胺酸酯激酶(ArgB)合成為N-乙醯基麩胺醯基磷酸酯，其消耗作為輔酶之ATP來產生ADP，且可為由argB基因編碼者。由於已知受到終產物L-精胺酸之反饋抑制，因此解除L-精胺酸之反饋抑制之修飾為已知，且已有報告指出使用此方式可改良L-精胺酸產生力(中國專利案案號102021154，與Amino Acids.2012 Jul；43(1)：255-66.doi：
10.1007/s00726-011-1069-x.Epub 2011 Sep 8)。
N-乙醯基麩胺醯基磷酸酯還原酶(ArgC)在大腸桿菌(E.coli)或酵母中亦稱為乙醯基麩胺酸酯半醛脫氫酶，且可由argC基因編碼。N-乙醯基麩胺醯基磷酸酯利用此酵素轉化成N-乙醯基麩胺酸酯5-半醛。使用NADPH作為供應能量之輔酶。所產生之N-乙醯基麩胺酸酯5-半醛使用L-麩胺酸酯作為胺基酸供體，轉化成N-乙醯基鳥胺酸，此反應係由乙醯基鳥胺酸胺基轉化酶(ArgD)所介導。乙醯基鳥胺酸胺基轉化酶可由argD基因編碼。所轉化之N-乙醯基鳥胺酸利用麩胺酸酯N-乙醯基轉化酶(ArgJ)之再循環反應，將其乙醯基遞送給L-麩胺酸酯，並呈L-鳥胺酸進行反應。
鳥胺酸胺甲醯基轉化酶(ArgF)通常稱為鳥胺酸胺甲醯化酶，且可由argF或argF2基因編碼。L-鳥胺酸結合胺甲醯基磷酸酯，形成L-瓜胺酸，並產生磷酸酯作為反應副產物。所產生之L-瓜胺酸最後經由精胺基琥珀酸合成酶(ArgG)與精胺基琥珀酸裂解酶(ArgH)(其等與上述精胺酸操縱子分開存在)之酵素反應合成L-精胺酸。L-精胺酸係由一共8個生物合成步驟所合成，本發明中，藉由強化精胺酸操縱子(argCJBDFR)活性來誘發L-精胺酸生產力的增強。
構成精胺酸操縱子之酵素只要具有上述活性均可包括在本發明範圍內，明確言之，該等酵素可為衍生自棒狀桿菌屬微生物之蛋白質。更明確言之，麩胺酸酯
N-乙醯基轉化酶(ArgJ)可包括SEQ ID NO：19之胺基酸序列，或與該序列之同源性為至少70%，明確言之80%，更明確言之90%或更高之胺基酸序列。N-乙醯基麩胺酸酯激酶(ArgB)可包括SEQ ID NO：21之胺基酸序列，或與該序列之同源性為至少70%，明確言之80%，更明確言之90%或更高之胺基酸序列。此外，若使用相應之酵素，則可引進相關技藝已知之修飾，以解除精胺酸之反饋抑制。N-乙醯基麩胺醯基磷酸酯還原酶(ArgC)可包括SEQ ID NO：23之胺基酸序列，或與該序列之同源性為至少70%，明確言之80%，更明確言之90%或更高之胺基酸序列。乙醯基鳥胺酸胺基轉化酶(ArgD)可包括SEQ ID NO：25之胺基酸序列，或與該序列之同源性為至少70%，明確言之80%，更明確言之90%或更高之胺基酸序列。鳥胺酸胺甲醯基轉化酶(ArgF)可包括SEQ ID NO：1或SEQ ID NO：3之胺基酸序列，或可包括與胺基酸序列SEQ ID NO：1或SEQ ID NO：3之同源性為至少70%之胺基酸序列。明確言之，鳥胺酸胺甲醯基轉化酶(ArgF)可包括與胺基酸序列SEQ ID NO：1或SEQ ID NO：3之同源性為至少75%、80%、85%、90%、95%、96%、97%、98%、或99%或更高之胺基酸序列。此外，顯然該等包括刪除、修飾、取代、或增加一或多個胺基酸殘基之胺基酸序列，只要其與上述蛋白質具有同源性並與上述蛋白質具有實質上相同或相應之生物活性時，均屬於本發明範圍內。
本文所使用術語“同源性”係指比對兩種
胺基酸序列或核苷酸序列之間之相似性程度，並可裸視比對或使用生物資訊演算法(其可藉由排比要比對之序列，提供同源性程度之分析結果)，來決定其同源性。兩種胺基酸序列之間之同源性可使用百分比表示。可在Wisconsin Genetics Software Package套裝軟體(Genetics Computer Group,Madison,WI,USA)之GAP、BESTFIT、FASTA、與TFASTA電腦軟體模模組中使用適用之自動化演算法。其他適用之演算法與排比時之同源性測定法已可以軟體，如：FASTP、BLAST、BLAST2、PSIBLAST、與CLUSTAL W自動執行。
本發明中，精胺酸操縱子活性之加強可指精胺酸操縱子中所存在酵素中至少一種酵素之活性加強，然而，其中並不包括argR基因單獨的活性的單一加強。例如：加強精胺酸操縱子活性可指透過加強精胺酸操縱子中所存在一種酵素之啟動子來加強操縱子中所存在所有酵素之活性，明確言之，可指藉由加強N-乙醯基麩胺醯基磷酸酯還原酶之啟動子來加強整個操縱子之活性。此外，本發明中，編碼構成精胺酸操縱子之酵素中至少一種酵素之基因之表現的增加亦可視為精胺酸操縱子活性的加強。
本文所使用術語“加強”活性係指使沒有蛋白質特定活性之微生物具有該蛋白質活性，或在具有該蛋白質活性之微生物中增加其細胞內活性等等，且係指使其細胞內蛋白質活性比內因性蛋白質活性增加。本文所使用術語內因性活性係指屬於棒狀桿菌屬之微生物之天然或
改造前狀態所具有之酵素活性態。
可採用相關技藝上已知之各種不同方法來加強或增加酵素活性。該等方法之實例雖然沒有限制，但可包括藉由進一步將包括編碼相應酵素之核苷酸序列之多核苷酸插入至染色體中或將該多核苷酸引至載體系統中等等來增加編碼該酵素之核苷酸序列之複本套數之方法、以強啟動子來置換酵素啟動子之方法，以及明確言之，可包括在啟動子上引進修飾之方法、及藉由基因修飾法將酵素改造成具有強活性者之方法。
本發明之明確實例可包括經由啟動子之修飾或取代來改造精胺酸操縱子中所存在酵素啟動子成為比內因性啟動子更強之啟動子之方法。具有核苷酸取代修飾之改良啟動子或異源性啟動子可取代內因性酵素之啟動子進行連接，該等異源性啟動子實例可包括pcj7啟動子(韓國專利案案號10-0620092)、lysCP1啟動子(韓國專利案案號10-0930203)、EF-Tu啟動子、groEL啟動子、aceA啟動子、aceB啟動子等等但不受此等限制。
本文所使用術語“啟動子”係指編碼區之非編碼核酸序列上游，其包括聚合酶結合區，並具有啟動轉錄形成啟動子基因下游之mRNA之活性，即，讓聚合酶結合並啟動該基因轉錄之DNA區，其位於mRNA轉錄起始區之5’區。
本發明中，可使用相關技藝上已知之各種不同方法加強鳥胺酸胺甲醯基轉化酶活性，且此等方法可
與上述相同。明確言之，可藉由將包括編碼該鳥胺酸胺甲醯基轉化酶之多核苷酸之表現載體轉形至細菌菌株內來達成該加強，但不受此等方法限制。
本文所使用術語“轉形”係指將DNA引至宿主內，藉以使所插入之DNA可如染色體外因子般可以複製，或採用染色體整合法使所插入之DNA可以複製。明確言之，可將本發明轉形體經由核酸分子序列之間之同源重組法插入染色體中，在該包括上述DNA之載體轉形至宿主細胞內後，在該載體中具有啟動子活性，並具有內因性標靶基因之啟動子區之序列，或可維持為質體形式。
本發明載體轉形之方法可包括可將核酸引至細胞內之任何方法，並可依據各宿主細胞選擇相關技藝上已知之任何適當標準技術並執行。可採用例如：電穿孔法、磷酸鈣(CaPO4)沉澱法、氯化鈣(CaCl2)沉澱法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚醣法、陽離子性脂質體法、乙酸鋰/DMSO法等等，但該等方法不受此等限制。
本文所使用術語“棒狀桿菌屬微生物(Corynebacterium sp.)”可指所有屬於具有產生L-精胺酸能力之棒狀桿菌屬之菌株，例如：麩胺酸棒狀桿菌(Corynebacterium glutamicum)、產氨棒狀桿菌(Corynebacterium ammonjagenes)、熱產氨棒狀桿菌(Corynebacteriumthermoaminogenes)、黄色短桿菌(Brevibacterium flavum)、發酵短桿菌(Brevibacterium fermentum)等等，但不受此等限制。明確言之，可使用麩
胺酸棒狀桿菌，但該等微生物不受此等限制。
另一態樣中，本發明提供一種產生L-精胺酸之方法，其包括在適當培養基中培養可產生L-精胺酸之棒狀桿菌屬微生物。
本發明中，該微生物培養法可依據相關技藝上習知方法進行，並可適當調整培養溫度、培養時間、培養基pH等條件。該等已知培養法詳細說明於參考文獻(Chmiel；Bioprozesstechnik 1.Einfuhrung in die Bioverfahrenstechnik(Gustav Fischer Verlag,Stuttgart,1991)，及Storhas；Bioreaktoren und periphere Einrichtungen(Vieweg Verlag,Braunschweig/Wiesbaden,1994))。此外，培養方法可包括批式培養法、連續培養法、與饋料-批式培養，明確言之，可採用批式過程或饋料-批式過程或重複饋料-批式過程進行連續培養，但不受此等限制。
所使用之培養基應適當符合特定菌株之需要條件。用於各種不同微生物之培養基係已知者(例如：來自美國細菌學會(American Society for Bacteriology)之“Manual of Methods for General Bacteriology”(Washington D.C.,USA,1981))。含在培養基中之碳源可包括醣類與碳水化合物(例如：葡萄糖、蔗糖，乳糖、果糖、麥芽糖、糖蜜、澱粉與纖維素)、油類與脂肪(例如：大豆油、葵花子油、花生油、與椰子油)、脂肪酸(例如：棕櫚酸、硬脂酸、與亞油酸)、醇類(例如：甘油與乙醇)、有機酸類(例如：乙酸)等等。此等材料可以單獨使用或呈混合物使用，但不受此
等限制。含在培養基中之氮源可包括含氮有機化合物(例如：蛋白腖，酵母抽出物、肉汁、麥芽抽出物、玉米漿液、大豆粉、與尿素)、及無機化合物(例如：硫酸銨、氯化銨、磷酸銨、碳酸銨、與硝酸銨)，此等材料可以單獨使用或呈混合物使用，但不受此等限制。含在培養基中之磷源可包括磷酸二氫鉀或磷酸氫二鉀或其等效之含鈉鹽，但不受此等限制。培養基可包含生長必要之金屬鹽(例如：硫酸鎂或硫酸鐵)，除了上述物質外，亦可使用促進生長之必要物質，如：胺基酸與維生素。此外可進一步添加適當前體至培養基中。上述之供應材料可一次加至培養基中或可在培養期間適當添加。
可使用鹼性化合物(例如：氫氧化鈉、氫氧化鉀、或氨)或酸性化合物(例如：磷酸或硫酸)適當調整培養基之pH。
可使用消泡劑(如：脂肪酸聚二醇酯)調整起泡性。可將氧氣或含氧氣體混合物(例如：空氣)引入培養基中，維持好氧條件。培養溫度可為20℃至45℃，明確言之，25℃至40℃。可連續培養至得到所需L-胺基酸之最大產量為止，明確言之，10小時至160小時。L-精胺酸可釋入培養基中或可保留在細胞中。
同時，包括培養上述微生物之本發明產生L-精胺酸方法可進一步包括在培養期間回收L-精胺酸之步驟。亦即本發明產生L-精胺酸之方法可包括於培養基中培養棒狀桿菌屬微生物，及從微生物與培養基中回收L-精胺
酸。該回收精胺酸之步驟可意指採用相關技藝上習知方法回收精胺酸之方法，從細胞或培養基中分離精胺酸。回收L-精胺酸之方法可包括離心、過濾、萃取、噴霧、乾燥、蒸發、沉澱、結晶、電泳、分段溶解、層析(例如：離子交換、親和力、疏水性、粒徑篩析、與高效液相層析法)等等，但不受此等限制。
根據本發明，利用具有加強精胺酸操縱子與鳥胺酸胺甲醯基轉化酶(ArgF或ArgF2)活性之產生L-精胺酸之棒狀桿菌屬微生物，可以高產量產生L-精胺酸。此外，該等以高產量產生之L-精胺酸可有效用於人類醫藥與製藥。
下文中，本發明將參考下列實例更詳細說明。然而，此等實例僅供說明目的，本發明並無意受此等實例限制。
為了加強微生物染色體上之精胺酸操縱子，構築一種載體，其中刪除N-乙醯基麩胺醯基磷酸酯還原酶(ArgC)之自我啟動子，並以不同啟動子取代。作為取代啟動子，使用lysCP1(SEQ ID NO：18，揭示於韓國專利案案號10-
0930203)，其具有強力誘發表現之活性。
首先，擴增DNA片段，其係經由初級聚合酶連鎖反應(PCR)，使用麩胺酸棒狀桿菌野生型菌株(登錄號：ATCC13869)之染色體DNA作為模板，及使用一對引子SEQ ID NO：13(SF_pargC_PR_pDC輸注引子(infusion primer)；5'-CGAGCTCGGTACCCGGGCAAAGAATACGGCTTCCTTGGC-3')與SEQ ID NQ：14(SR_pargC_PR_XbaI-XhoI-BamHI輸注/限制酶引子；5'-CTGGATCCTCGAGTCTAGAGACGGGTTAGACATGCAAAA-3')與一對引子SEQ ID NO：15(SF_pargC_PR_SpeI-ScaI-BamHI輸注/限制酶引子；5'-GACTCGAGGATCCAGTACTAGTATGATAATCAAGGTTGCAAT-3')與SEQ ID NO：16(SR_pargC_PR_pDC輸注引子；5'-TGCAGGTCGACTCTAGGGTAACGCCTTCTTTCAAAG-3')。PCR反應之明確條件如下：PCR反應之進行係使用PCR裝置(Bio-rad C1000熱循環器(thermal cycler))與Pfu聚合酶(Macrogen)，於95℃變性10分鐘，於55℃黏合30秒，及於72℃延長1分鐘，並重複28次循環。
所得初級PCR片段利用片段DNA純化套組(GeneAll)純化後，取三個DNA片段與pD載體(其已使用XmaI-XbaI限制酶分解而製成)混合而連接。所連接之DNA片段可使用In-fusion Cloning Kit選殖套組(Clontech)於50℃反應10分鐘，藉以構築成pD-RargC_PR載體。
該取代啟動子之插入係以lysCP1啟動子擴
增的方式進行，其係使用pDZ-lysCP1(韓國專利案案號10-0930203)作為模板，及使用一對引子SEQ ID NO：5(SF_PlysCP1_XhoI-XbaI輸注引子；5'-CCGTCTCTAGACTCGAGCCATCTTTTGGGGTGCGG-3')與SEQ ID NO：6(SR_PlysCP1_SpeI輸注引子；5'-TTGATTATCATACTAGTCTTTGTGCACCTTTCGAT-3')，其等與pD-PargC_PR載體(其係使用XhoI-SpeI限制酶分解而製成)混合而連接。依上述進行相同PCR與In-fusion Cloning選殖法，最後透過此等方法構築成pD-PargC：：lysCP1載體。
為了加強鳥胺酸胺甲醯基轉化酶(其係一種精胺酸生物合成酶)，構築一種重組體表現載體。使用p117-cj7-GFP(韓國專利案案號10-0620092)作為模板載體，使用EcoRV-XbaI限制酶處理排除該模板載體中編碼GFP之核苷酸序列，插入argF(其係衍生自麩胺酸棒狀桿菌野生型菌株ATCC13869)與argF2(韓國專利案案號10-0830290)。
擴增argF基因之DNA片段，其係經由PCR，使用麩胺酸棒狀桿菌野生型菌株(登錄號：ATCC13869)之染色體DNA作為模板，並使用一對引子SEQ ID NO：7(SF_argF_EcoRV輸注引子；5'-ACGAAAGGAAACACTCGATATCATGACTTCACAACCACAGGT-3')與SEQ ID NO：8(SR_argF_XbaI輸注引子；5'-GCCAAAACAGCTCTAGATTACCTCGGCTGGTGGGCCA-3'
)。PCR反應係使用Pfu聚合酶進行，於95℃變性10分鐘，於55℃黏合30秒，及於72℃延長2分鐘，並重複28次循環。所得PCR片段經過純化，與p117-cj7-GFP(其已經過EcoRV-XbaI限制酶處理)混合，採用In-fusion Cloning選殖法連接，藉以構築成重組體表現載體p117-Pcj7-argF。
該argF2基因之擴增係經由PCR，使用麩胺酸棒狀桿菌野生型菌株(登錄號：ATCC13032)之染色體DNA作為模板，並使用一對引子SEQ ID NO：9(SF_argF2_EcoRV輸注引子；5'-ACGAAAGGAAACACTCGATATCATGGCCAGAAAACATCTGCT-3')與SEQ ID NO：10(SR_argF2_XbaI輸注引子；5'-GCCAAAACAGCTCTAGACTACGCATTGATCGACCGAG-3')與Pfu聚合酶(Macrogen)，經由PCR，使用Pfu聚合酶，於95℃變性10分鐘，於55℃黏合30秒，及於72℃延長2分鐘，並重複28次循環。所得PCR片段經過純化，與p117-cj7-GFP(其已經過EcoRV-XbaI限制酶處理)混合，採用In-fusion Cloning選殖套組連接，藉以構築成重組體表現載體p117-Pcj7-argF2。
另外，構築一種可同時表現argF與argF2兩種基因之重組體表現載體。所構築之表現載體p117-Pcj7-argF經過NotI處理後，取p117-Pcj7-argF2插入至其中。明確言之，進行PCR反應，其係使用重組體質體p117-Pcj7-argF2作為模板，並使用SEQ ID NO：11(SF_Pcj7_argF2_NotI輸注引子；
5'-CCTTTTTGCGGCGGCCGCAGAAACATCCCAGCGCTACT-3')與SEQ ID NO：12(SR_argF2_NotI輸注引子；5'-CACCGCGGTGGCGGCCGCCGCAAAAAGGCCATCCGTCA-3')引子與Pfu聚合酶，於95℃變性10分鐘，於55℃黏合30秒，及於72℃延長2.5分鐘，並重複28次循環。所得PCR片段經過純化，與p117-Pcj7-argF(其已經過NotI限制酶處理)混合，並採用In-fusion Cloning選殖套組連接，最後構築成重組體表現載體p117-Pcj7-argF/Pcj7-argF2。
為了取代棒狀桿菌染色體上之精胺酸操縱子之自我啟動子，將實例1所構築之重組體載體pD-PargC：：lysCP1轉形至現有之產生精胺酸之棒狀桿菌屬菌株中，藉以構築成已插入重組體載體之棒狀桿菌屬菌株。明確言之，藉由將實例1所構築之重組體載體pD-PargC：：lysCP1轉形至現有之生產精胺酸之菌株KCCM10741P(韓國專利案案號10-07916590)與ATCC21831，使lysCP1啟動子序列插入染色體中，藉此使該母菌株所擁有之自我啟動子序列透過同源性重組被該載體之啟動子序列所取代。
進行轉形時，採用電脈衝法先將該重組體載體插入KCCM10741P與ATCC21831中(Appl Microbiol Biotechnol.1999 Oct；52(4)：541-5)，在含25mg/L卡納黴素(kanamycin)之培養基中選拔該已藉由同源性序列重組插入
其染色體之菌株。所選拔之初級菌株經過交叉處理(cross-over)，藉以選拔彼等已改用lysCP1啟動子取代啟動子並已排除載體之菌株。藉由PCR，使用一對引子SEQ ID NO：5與SEQ ID NO：6確認最終轉形菌株中存在該取代之啟動子，該等菌株稱為KCCM10741P_△ PargC：：lysCP1與ATCC21831_△ PargC：：lysCP1。
將實例2所構築之重組體表現載體：p117-Pcj7-argF、p117-Pcj7-argF2、與p117-Pcj7-argF/Pcj7-argF2，採用電脈衝法，插入至菌株KCCM10741P_△ PargC：：lysCP1與ATCC21831_△ PargC：：lysCP1中，於包含25mg/L卡納黴素(kanamycin)之培養基中選拔，最後構築進一步表現argF、argF2、與argF/argF2之菌株。該等菌株稱為KCCM10741P_△ PargC：：lysCP1_Pcj7-argF，KCCM10741P_△ PargC：：lysCP1_Pcj7-argF2，KCCM10741P_△ PargC：：lysCP1_Pcj7-argF/Pcj7-argF2，ATCC21831_△ PargC：：lysCP1_Pcj7-argF，ATCC21831_△ PargC：：lysCP1_Pcj7-argF2，與ATCC21831_△ PargC：：lysCP1_Pcj7-argF/Pcj7-argF2，且其中KCCM10741P_△ PargC：：lysCP1_Pcj7-argF2稱為CA06-2044，並依據布達珮斯條約(Budapest Treaty)，於2013年12月9日寄存在韓國微生物保存中心(Korean Culture Center of Microorganisms)(KCCM)，登錄號KCCM11498P。
使用以下麩胺酸棒狀桿菌檢測加強精胺酸操縱子與鳥胺酸胺甲醯基轉化酶對產生精胺酸之能力之影響：KCCM10741P_△ PargC：：lysCP1，KCCM10741P_△ PargC：：lysCP1_Pcj7-argF，KCCM10741P_△ PargC：：lysCP1_Pcj7-argF2，KCCM10741P_△ PargC：：lysCP1_Pcj7-argF/Pcj7-argF2，ATCC21831_△ PargC：：lysCP1,ATCC21831_△ PargC：：lysCP1_Pcj7-argF，ATCC21831_△ PargC：：lysCP1_Pcj7-argF2，與ATCC21831_△ PargC：：lysCP1_Pcj7-argF/Pcj7-argF2，其等係實例3所構築之產生精胺酸之菌株，其培養係如下所示。特定言之，使用麩胺酸棒狀桿菌KCCM10741P與ATCC21831，其為母菌株，係作為對照組，分別取一白金環之菌株接種至含25mL(6%葡萄糖、3%硫酸銨、0.1%磷酸鉀、0.2%硫酸鎂七水合物、1.5%玉米漿液(CSL)、1% NaCl、0.5%酵素抽出物、與100μg/L生物素，pH 7.2)生產培養基之250mL帶有角擋板的燒瓶中，於30℃與200rpm培養48小時。培養完成時，以HPLC測定L-精胺酸產量，結果示於下表1。
如上表1所示，其中編碼精胺酸操縱子與鳥胺酸胺甲醯基轉化酶之基因被同時加強之菌株所顯示產生精胺酸之能力比對照組最大提高50%。此外，精胺酸濃度與鳥胺酸濃度提高顯示於僅加強精胺酸操縱子(KCCM10741P_△ PargC：：lysCP1與
ATCC21831_△ PargC：：lysCP1)時，藉由引進argF、argF2或argF與argF2來解析，最後結果顯示精胺酸濃度提高。
習此相關技藝者可由上文中了解本發明可在不修改本發明之技術觀念或基本特徵下具體實施。因此本文所揭示之具體實施例僅供說明之目的，不應視為本發明之限制範圍。反之，本發明不僅涵蓋該等具體實施例，而且亦涵蓋各種不同替代、修飾、等效及其他具體實施例，其等均包括在附錄之申請專利範圍所界定之本發明本質與範圍內。
1.中華民國、財團法人食品工業發展研究所、2015年12月4日、BCRC910717
1.大韓民國、韓國微生物保存中心、2013年12月09日、KCCM11498P
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Claims (4)
- 一種具有產生L-精胺酸之能力之棒狀桿菌(Corynebacterium)屬微生物，其具有加強之精胺酸操縱子與鳥胺酸胺甲醯基轉化酶活性。
- 如申請專利範圍第1項所述之微生物，其中該鳥胺酸胺甲醯基轉化酶為如SEQ ID NO：1或SEQ ID NO：3代表之胺基酸序列。
- 如申請專利範圍第1項所述之微生物，其中該棒狀桿菌屬微生物為麩胺酸棒狀桿菌(Corvnebacterium glutamicum)。
- 一種產生L-精胺酸之方法，其包括：於培養基中培養如申請專利範圍第1至3項中任一項所述之棒狀桿菌屬微生物；及從該微生物或培養基中回收L-精胺酸。
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2015
- 2015-05-29 KR KR1020150076331A patent/KR101835935B1/ko active IP Right Grant
- 2015-10-13 RU RU2016147322A patent/RU2671106C1/ru active
- 2015-10-13 ES ES15850829T patent/ES2724000T3/es active Active
- 2015-10-13 BR BR112016029730-0A patent/BR112016029730B1/pt active IP Right Grant
- 2015-10-13 HU HUE15850829A patent/HUE042618T2/hu unknown
- 2015-10-13 JP JP2016575080A patent/JP6476212B2/ja active Active
- 2015-10-13 MY MYPI2017000069A patent/MY188962A/en unknown
- 2015-10-13 DK DK15850829.1T patent/DK3153573T3/da active
- 2015-10-13 TW TW104133479A patent/TWI583790B/zh active
- 2015-10-13 CN CN201580055711.1A patent/CN107002027B/zh active Active
- 2015-10-13 CA CA2951019A patent/CA2951019C/en active Active
- 2015-10-13 PL PL15850829T patent/PL3153573T3/pl unknown
- 2015-10-13 AU AU2015331160A patent/AU2015331160B2/en active Active
- 2015-10-13 US US15/515,551 patent/US10626426B2/en active Active
- 2015-10-13 EP EP15850829.1A patent/EP3153573B1/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| MY188962A (en) | 2022-01-14 |
| AU2015331160A1 (en) | 2017-01-05 |
| US10626426B2 (en) | 2020-04-21 |
| EP3153573A1 (en) | 2017-04-12 |
| BR112016029730A2 (pt) | 2017-10-24 |
| HUE042618T2 (hu) | 2019-07-29 |
| DK3153573T3 (da) | 2019-07-01 |
| CN107002027B (zh) | 2021-05-28 |
| CA2951019A1 (en) | 2016-04-21 |
| PL3153573T3 (pl) | 2019-08-30 |
| KR101835935B1 (ko) | 2018-03-12 |
| EP3153573A4 (en) | 2017-12-06 |
| US20170226545A1 (en) | 2017-08-10 |
| EP3153573B1 (en) | 2019-04-03 |
| JP2017518760A (ja) | 2017-07-13 |
| RU2671106C1 (ru) | 2018-10-29 |
| ES2724000T3 (es) | 2019-09-05 |
| KR20160043890A (ko) | 2016-04-22 |
| JP6476212B2 (ja) | 2019-02-27 |
| AU2015331160B2 (en) | 2018-08-02 |
| BR112016029730B1 (pt) | 2023-11-14 |
| CA2951019C (en) | 2021-11-16 |
| CN107002027A (zh) | 2017-08-01 |
| TWI583790B (zh) | 2017-05-21 |
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