質體含 aroY/aroZ 基因之建構

Construction of AroY/AroZ Encoded Plasmid

朱孝凱(Hsiao Kai Chu)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

己二酸的用途很廣，可用來生產各種塑料及纖維，如耐綸 6,6。己二酸可由生物發酵得到己二烯二酸再氫化而來。使用葡萄糖為原料生產己二烯二酸代謝途徑之中間產物鄰苯二酚對細胞具有毒性，與大腸桿菌 (Escherichia coli, E. coli)相較綠膿桿菌 (Pseudomonas putida)對鄰苯二酚的耐受性較佳，因此我們使用 Pseudomonas putida F1 作為生產己二烯二酸之菌種，由於Pseudomonas putida F1生產己二烯二酸的代謝途徑中缺少基因 aroY 和 aroZ，我們利用聚合酶連鎖反應 (polymerase chain reaction, PCR) 從克雷伯氏肺炎菌 (Klebsiella pneumonia) 的染色體中複製，並在前方設計適當的核糖體結合點（ribosome binding site, RBS），接上 catB 相同同源序列。之後利用電穿孔法將此基因片段送進 Pseudomonas putida F1，將 aroZ-aroY 以同源重組方式置換進入染色體中。CatB 蛋白的去除可阻止己二烯二酸被代謝進入檸檬酸循環，綠膿桿菌 catB基因本身的啟動子受到己二烯二酸濃度的正調控，當己二烯二酸濃度越高，越能表現該基因，如此將有助於 aroZ 及 aroY 之表現進而增加己二烯二酸之產率。

關鍵字

catBC 啟動子 ； 綠膿桿菌 ； 己二烯二酸

並列摘要

Adipic acid is the monomer for various plastics and fibers, especially nylon 6,6. Adipic acid is presently produced by oxidation of a mixture of cyclohexanol and cyclohexanone with nitric acid. It can also produced by bio-fermentation after hydrogenation of cis,cis-muconic acid. Catechol, one of the intermediates in the metabolic pathway, is usually toxic to cells. However, Pseudomonas putida is able to withstand a high concentration of catechol. Therefore, Pseudomonas putida is a good host for synthesis of muconic acid from glucose. To endow Pseudomonas putida with the capacity to produce cis,cis-muconic acid from glucose, we transformed aroZ (3-dehydroshikimate dehydratase) and aroY (protocatechuic decarboxylase) into Pseudomonas putida. aroZ and aroY containing ribosome binding sites were separately amplified from Klebsiella pneumonia using polymerase chain reaction (PCR) and then were ligated into pTZ57R/T vector. Two 50-bp homologous sequences of catB were flanked to the aroZ-aroY fragment via PCR becoming aroZ-aroY-50. This aroZ-aroY-50 DNA fragment was then electro-transformed into Pseudomonas putida F1 and recombined with catB using the Red/ET recombination system. The chromosomal catBC promoter controls the transcription of aroZ-aroY and can be activated by cis,cis-muconic acid.

並列關鍵字

catBC promoter ； Pseudomonas putida ； cis,cis-muconic acid

參考文獻

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