- 學位論文
利用大腸桿菌表現巨大芽孢桿菌聚羥基烷酸合成基因之研究
Expression of polyhydroxyalkanoates synthesis genes from Bacillus megaterium BCRC10608 in Escherichia coli.
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摘要
聚羥基烷酸(polyhydroxyalkanates, PHAs)是微生物中在營養不平衡的條件下，將多餘的碳源轉換成脂質堆積於胞內之聚合物。巨大芽孢桿菌(Bacillus megaterium)具有堆積polyhydroxybutyrate(PHB)的能力，其合成PHAs之主要基因包括phaPQRBC，其中phaC在其合成機制中扮演聚合反應的角色。本研究目的在於選殖B. megaterium合成PHAs的這些相關基因，並將其於大腸桿菌DH5α內表現。實驗並以添加4% 葡萄糖、4% 琥珀酸鹽以及1% 甘油作為碳源，以探討其生成之產物的產率及物理性質。研究結果發現，B. megaterium於LB培養液中添加1% 甘油生長情況較佳，產物則為PHB。而在重組大腸桿菌方面，菌株EYT09(DH5α / pBluescript II SK (+)::phaRBC)可成功的表現經起始密碼子改變phaC之PhaC蛋白質(39.82 KDa)。另外在LB培養液添加1% 甘油培養下，重組大腸桿菌EYT09及EYT11 (DH5α / pBluescript II SK (+)::phaC)在氣相層析儀分析結果中，可發現於6.3分鐘中有一吸收鋒面的存在，產率分別為3.67%及11.50%；然而進一步分析發現此產物並非與PHAs相關之產物。雖然如此，本實驗仍然成功的在大腸桿菌內表現B. megaterium之PhaB與PhaC蛋白質。
並列摘要
Polyhydroxyalkanoates (PHA) are polyesters accumulated by various bacteria under unbalanced growth conditions when the carbon substrate is in excess to other nutrients such as nitrogen, sulfur, phosphorus or oxygen. B. megaterium, the first microorganism that has been discovered for the ability to accumulate intracellular polyhydroxybutyrate (PHB), was demonstrated to be able to accumulate polyhydroxyvalerate (PHV) when supply the bacterium with certain carbon source (e.g. sodium succinate). Biosynthesis of PHB in B. megaterium involves genes including phaPQRBC. The ability and mechanisms (and genes involved) in PHV synthesis in this bacterium have not been described before. In this study, we have examined the production of PHB and PHV by B. megaterium using different carbon sources. Production of PHB in recombinant Escherichia coli harboring the B. megaterium PHB synthesis genes (phaP, phaQ, phaR, phaB and phaC) was also evaluated. Here we attempt to clone these genes into suitable E. coli host in order to (1) construct a recombinant strain of E. coli using PHB synthesis genes of B. megaterium to produce PHB; (2) examine if PHV (or other PHA) can be III produced in the recombinant Escherichia coli when supplemented with different carbon sources. The results of thesis shown that B. megaterium was able to produced PHB when grown with a variety of carbon sources including glycerol. Expression of PhaB and PhaC protein can be detected in the recombinant Escherichia coli ETY09 (DH5α / pBluescript II SK (+)::phaRBC) and EYT11 (DH5α / pBluescript II SK (+)::phaC). However, there was no detectable polyhydroxyalkanoates in these recombinant strains by Gas chromatography and FTIR. Some other gene(s) of B. megaterium might be required in order to produce PHB in the recombinant E. coli and this remained to be investigated.
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