- 作者: 黃克榮; 許宗雄
- 作者服務機構: 國立清華大學生命科學研究所
- 中文摘要:
天冬胺酸激?催化天冬胺酸磷酸化。在大腸桿菌的代謝途徑中,對離胺酸敏感之天冬胺酸激?Ⅲ為控制離胺
酸生合成的一個重要酵素,已知其酵素活性及基因的表現會受終產物離胺酸的迴饋控制。為探討大腸菌中此酵素基因
lysC的放大表現對離胺酸生合成的影響,本研究乃利用性狀互補方式選殖到不含起動子的lySC基因,並以pUC19當載
體構成質體pUC19-AK3,此質體以乳糖起動子lac負責基因的表現,但保有原基因的核醣體結合部位與轉錄終止信
號,此質體於大腸菌DH1菌株中的基因表現不受離胺酸影響,但過度表現的產物酵素,其酵素活性仍會受到離胺酸及白
胺酸的控制,進一步SDS聚丙烯醯胺電泳分子量測定與N-端胺基酸序列分析,顯示此表現的產物酵素與原天冬胺酸激
?Ⅲ相同,此重組菌株DH1/pUC19AK3於對數生長後期的基本培養液中累積離胺酸濃度約為7mM,而原DH1菌株累
積離胺酸濃度則偵測不到,顯示天冬胺酸激?Ⅲ在菌體內濃度的增加能有效的增加培養液中離胺酸的累積濃度,另外,
天冬胺酸激?Ⅲ在菌體內濃度的增加亦會影響其它非胺基酸代謝物濃度的變化,但影響較小,將重組菌株DH1/pUC19
AK3於對數生長後期的菌體收集再轉培養於新鮮培養液,如培養液中添加有天冬胺酸,則可進一步大量提高培養液中生
產的離胺酸濃度,與未添加有天冬胺酸的對照比較,添加0.2%天冬胺酸的培養液,於45分鐘可達最高的約55mM離胺酸
濃度,約八倍於未添加有天冬胺酸者。
e - 英文摘要: A promoterless lysC gene, coding for Escherichia coli aspartokinase III (AKase III), has been clonedby phenotypic complementation using plasmid pUC19 as the vector. The hybrid plasmid obtained, pUC19AK3,preserved the ribosome binding site and transcriptional termination signal of the gene but with a lac pro-moter. E. coli strains containing the recombinant plasmid had high levels of AKase III activity. AKase IIIactivity from expressing strains was inhibited by lysine, leucine, and S-(2-aminoethyl)-L-cysteine (AEC)but not by threonine and methionine. The overexpressed AKase III enzyme had a molecular weight ofabout 50 kD from SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis con-firmed that the product from the hybrid plasmid was identical to native AKase III rather than a fusionprotein. Moreover, overexpression of AKase III significantly increased lysine excretion in the plasmid-harboring E. coli strain DH 1. This increase in the level of AKase III activity also affected other metabolitesthan lysine. Addition of aspartate to the medium brought about significant increases in lysine excretion.A maximum increase (about 8-fold) in lysine accumulation was observed 45 minutes after incubation inminimal medium containing 0.2% aspartate as compared to aspartate-free medium.
- 中文關鍵字: aspartokinase; Escherichia coli; lysine; gene cloning; promoters
- 英文關鍵字: --