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標題: 利用重組大腸桿菌異源表現極端高鹽太古生物聚羥基烷酯之研究
Heterologous expression of polyhydroxyalkanoate from extreme halophilic archaeon Haloterrigena sp. H13 in Escherichia coli
作者: 陳禹安
Chen, Yu-An
關鍵字: extreme halophilic archaeon;極端高鹽太古生物;polyhydroxyalkanoate;聚羥基烷酯
出版社: 生命科學系所
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聚羥基烷酯(polyhydroxyalkanoate, PHA)為一種微生物型聚酯,當微生物面臨營養不平衡狀態時,會在胞內以疏水性的包涵體累積PHA在胞內,是一具有生物相容及生物降解等特性的熱塑性聚合物,因此在醫學與工業上有良好的應用性。由高鹽太古生物Haloterrigena sp. H13所生產的新型聚酯(hPHA)具有特殊的不飽和乙炔基(C≡C);經由細胞毒性與貼附測試,結果顯示hPHA對細胞沒有毒性,且細胞相容性與貼附性均優於細菌型的polyhydroxybutyrate (PHB),具有作為生醫材料的潛力。Haloterrigena sp. H13的PHA生合成基因群組由maoC、phaR、phaT、phaD、phaC和phaB等基因組成。利用大腸桿菌異源表現的Haloterrigena sp. H13 PHA聚合酶進行活性測試,顯示需同時存在PhaCH13和PhaDH13兩個蛋白單元才會具有聚合PHA的活性。此研究先以南方墨漬法與基因序列分析,證實Haloterrigena sp. H13的PHA生合成基因群組在phaBH13下游具有三個反向的基因,分別為npdH13、moaCH13、yjeFH13,並沒有如一般細菌所具有的phaA。由Haloterrigena sp. H13的PHA生合成基因群組成員,可推測PHA生合成可能經由MaoCH13將脂肪酸代謝的中間產物轉化成(R)-hydroxyacyl-CoA,再由PhaDH13、PhaCH13聚合成PHA。因而將hPHA生合成基因phaCH13、phaDH13以及PHA單體提供者基因maoCH13構築在大腸桿菌LS5218這株脂肪酸代謝過度表現株中,來利用重組大腸桿菌LS5218-pTrDCMH13快速生產hPHA。基因轉錄與蛋白表現分析,皆顯示phaCH13、phaDH13與 maoCH13確能在大腸桿菌LS5218轉錄與轉譯,且具有異源表現phaCH13與phaDH13的大腸桿菌LS5218亦具有PHA聚合酶酵素活性。但以蘇丹黑染色和尼羅河紅染色及氣相層析儀PHA單體分析與定量,都無法證實重組大腸桿菌胞內具有PHA顆粒。

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria and Archaea when facing the nutrient-limit stress and with excess carbon source. PHA is a kind of thermoplastic polymer with biocompatible and biodegradable characteristics, therefore has good medical and industrial applications. Extrem halophilic archaeon Haloterrigena sp. H13 produced a new type of unsaturated polyester with acetylenic linkage (C ≡ C). After cytotoxicity and cells adhesion test showed hPHA are non-toxic, biocompatibility and attachment are better than polyhydroxybutyrate (PHB), has the potential for biomedical materials. PHA biosynthesis genes cluster in Haloterrigena sp. H13 consist of maoCH13, phaRH13, phaTH13, phaDH13, phaCH13 and phaBH13. PHA activity assay tests indicate heterologous expressional PHA synthase has activity only when exist PhaCH13 and PhaDH13. In this studies, using Southern blot and gene sequence analysis, confirmed PHA biosynthesis genes cluster of Haloterrigena sp. H13. Results showed that three reverse genes except phaA at downstream of phaBH13 are npdH13, moaCH13, yjeFH13, respectively. Based on the PHA biosynthesis genes cluster members of Haloterrigena sp. H13, we suggest that PHA biosynthesis pathway of Haloterrigena sp. H13 may be catalyst the intermediates from fatty acid metabolism into (R)-hydroxyacyl-CoA by MaoCH13 then polymerized into PHA by PHA synthase (PhaDH13 and PhaCH13). Thus construct PHA synthetic pathway of Haloterrigena sp. H13 by heteologous expressed phaDH13, phaCH13, and maoCH13 into E. coli fadR defective strain LS5218 for rapid accumulated PHA. Transcriptional test and protein expression analysis are showed phaCH13, phaDH13 and maoCH13 can really transcribed and translated in E. coli LS5218, and heterologous expressional PhaCH13 PhaDH13 in E. coli LS5218 also showed PHA synthase activity. However, PHA granule did not detected in E. coli LS5218-pTrDCMH13 either by Sudan black or Nile red staining.
其他識別: U0005-0902201111185600
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