• 作者： 蘇遠志; 簡相堂
• 作者服務機構： 國立臺灣大學農業化學研究所
• 中文摘要： 本研究之目的為利用基因破壞與置換技術，封閉蘇胺酸及甲硫胺酸之生合成路徑，由此構築具有穩定遺傳性狀之
  Brevibacterium divaricatum離胺酸生產菌株。利用聚合?鏈鎖反應由B. divaricatum之染色體中合成1.8，1.25及2.8 kb之
  hom, thrB和hom-thrB基因片段，並選殖於大腸桿菌一棒狀桿菌之穿梭載體pSUMN 18中。將質體pUC4-KISS中之kana-
  mycin耐性基因插入上述基因中加以破壞，並以此構築嵌入質體，再轉形至B. divaricatum菌株中篩選嵌入轉形株。結果
  顯示B. divaricatum具有強烈之宿主鑑識屏障，惟有由避開此屏障之DNA構築之嵌入質體始能獲得嵌入轉形株。此等轉
  形株皆具kanamycin耐性且無任何質體。其中約有1一10％具營養要求性，經由檢測，皆為需求蘇胺酸和∕或高絲胺酸
  者，與預期相符。南方氏漬染分析證實其為嵌入轉形株，推測可能具有單一和雙重互換之基因重組形式。此類營養要求
  轉形株在限制蘇胺酸供應之條件下，可於培養液中蓄積1一3％之離胺酸。其中以破壞hom或hom-thrB者較破壞thrB者為
  佳。此類轉形株之遺傳性狀極為穩定，其回復變異頻率每世代低於10 。
  o
• 英文摘要： Gene disruption and replacement techniques were applied to block the biosynthesis of threonine andmethionine and thus to construct genetically stable lysine producers in a glutamate-producing bacteria,Brevibacterium divaricatum. The homoserine dehydrogenase gene (hom), homoserine kinase gene (thrB)and hom-thrB operon were amplified as 1.8, 1.25 and 2.8 kb fragments from B. divaricatum by polymerasechain reaction (PCR) and cloned in an E. coli-coryneform bacteria shuttle vector, pSUMN18. These geneswere disrupted by inserting a kanamycin resistant gene (kan) from pUC4-KISS into the structural genes. Integrative plasmids were constructed and transformed into B. divaricatum. Integrative transformants couldbe obtained only when the integrative plasmids were constructed from the plasmids which had escaped fromthe restriction barrier of the hosts. The resulting integrative transformants showed kanamycin resistanceand contained no plasmids. About 1-10 % of the transformants were auxotrophs. By checking the nutri-tional requirement, it was found that all of these transformants required threonine and/or homoserineas expected. Southern blot analysis confirmed the integrations, and both single and double crossover homo-logous recombination mechanisms were proposed to explain the integration and replacement. These auxo-trophic integrative transformants which were derived from double crossover events accumulated 1-3％lysine in culture broth only when the added threonine was limited. Integrative transformants which weresite-specifically inactivated in hom or hom-thrB genes produced more lysine than did those only inactivatedat the thrB gene. These transformants were extremely stable, and the reversion frequency was below 10 per generation. It is suggested that this technique will be useful in the construction of stable auxotrophicmutants.
• 中文關鍵字： Brevibacterium divaricatum; gene disruption and replacement; genetic stability; DNA restric-tion; hom gene; homologous recombination; hom-thrβ genes; L-Lysine production; polymerase chain reaction; thrβ gene.
• 英文關鍵字： --