Lee S Y, Yim K S, Chang H N, Chang Y K
BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yusung-gu, Taejon.
J Biotechnol. 1994 Feb 14;32(2):203-11. doi: 10.1016/0168-1656(94)90183-x.
Plasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure. Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures. Higher instability was observed when cells were accumulating PHB. Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture. Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively. They showed 100% stability even during PHB synthesis and accumulation over 110 generations. All four plasmids were structurally stable. The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 101.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104). The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.
构建了含有嗜碱假单胞菌聚（3-羟基丁酸）（PHB）生物合成基因的质粒，用于在大肠杆菌中生产PHB，并通过在无抗生素压力下反复传代培养来研究质粒稳定性。pSYL101（高拷贝）和pSYL102（中拷贝）在传代培养过程中均不稳定。当细胞积累PHB时，观察到更高的不稳定性。在补料分批培养过程中，无质粒细胞的快速生长以及含有大量PHB的不分裂细胞的出现加剧了分离不稳定性。然后通过分别将质粒R1的parB基因座克隆到pSYL102和pSYL101中，开发了两种衍生物pSYL103和pSYL104。即使在PHB合成和积累超过110代的过程中，它们也显示出100％的稳定性。所有四种质粒在结构上都是稳定的。通过XL1-Blue（pSYL104）的高细胞密度培养，在39小时内分别获得了最终细胞质量、PHB浓度和每干细胞重量的PHB（P/X，w/w，％），分别为101.4 g l-1、81.2 g l-1和80.1％。使用XL1-Blue（pSYL103）时最终PHB浓度较低，这表明在大肠杆菌中，PHB的合成和积累到高浓度需要高基因剂量。
