【摘要】 为实现重组大肠杆菌以葡萄糖为唯一碳源合成均聚的P( 4HB) ,PCR扩增大肠杆菌编码谷氨酸:琥珀酰半缩醛转氨基酶基因(gabT) ,谷氨酸脱羧酶基因(gadA)以及富养罗尔斯通氏菌(Ralstoniaeutropha)H16的4_羟基丁酸脱氢酶基因(gadB) ,并组装到携带富养罗尔斯通氏菌(Ralstoniaeutropha)H16的PHA聚合酶基因(phaC)和克氏梭菌(Clostridiumkluyveri)中编码4_羟基丁酸:CoA转移酶基因(orfZ)的重组质粒pKESS5 3上,形成一个大的操纵元。携带重组质粒的大肠杆菌获得从三羧酸循环的中间物———α_酮戊二酸到P( 4HB)的代谢途径。结果表明,重组大肠杆菌可以以葡萄糖为唯一碳源合成均聚的P( 4HB) ,当向以葡萄糖为唯一碳源的无机培养基添加蛋白胨、酵母提取物、酪蛋白水解物时,P( 4HB)的含量可以高达菌体干重的30 %。更多还原

【Abstract】 The aim of this study is to construct recombinantEscherichia coli strains capable of producing poly(4_hydroxybutyric acid) homopolyester from glucose as sole carbon source. A glutamate:succinate semialdehyde transaminase gene fromEscherichia coli, a glutamate decarboxylase gene fromE. coli, and a 4_hydroxybutyrate dehydrogenase gene fromRalstonia eutrophawere cloned by PCR and assembled onto the plasmid pKSSE5.3 which haboured the PHA synthase gene fromRalstonia eutropha and 4_hydroxybutyrate:CoA transferase fromClostridium kluyveri. The resulting plasmids were transformed intoE. coliand the pathway for biosynthesis of poly(4_hydoxybutyric acid) from glucose via α_ketoglutarate, an intermediate in TCA cycle was established in recombinantE. colistrains. Recombinant strains synthesized the homopolyester P(4HB), when cells were cultivated in Luria_Bertani broth with glucose as carbon source. P(4HB) accumulation was enhanced up to 30% of cell dry weight, when cells were cultivated in mineral salts M9 medium plus glucose as sole carbon source with addition of yeast extract, tryptone, casein hydrolate into medium respectively.更多还原