Kalousek S, Lubitz W
Institute of Microbiology and Genetics, University of Vienna, Austria.
Can J Microbiol. 1995;41 Suppl 1:216-21. doi: 10.1139/m95-190.
The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria-Bertani medium without glucose supplement.
产碱杆菌的聚（β-羟基丁酸酯）（PHB）生物合成基因组织在一个单一操纵子（phbCAB）中，已被克隆到大肠杆菌中。在大肠杆菌中，当细胞单独在Luria-Bertani培养基中生长或分别添加1%葡萄糖（w/v）时，来自质粒p4A的野生型phb操纵子中基因的表达会导致形成占细胞干重10%或50 - 80%的PHB。为了在Luria-Bertani培养基中进一步刺激不依赖额外碳源的PHB形成，已应用分子方法为PHB合酶基因（phbC）提供有效的大肠杆菌转录和翻译信号。phbC序列上游存在的lac启动子使其表达能够根据所选宿主菌株的LacI状态进行控制。T7基因10核糖体结合位点用于翻译起始。对大肠杆菌中PHB的产生进行了比较，比较的菌株要么含有携带完整phbCAB操纵子的质粒p4A，要么含有两个兼容质粒，这两个质粒携带在lac启动子 - 操纵子区域转录控制下的β-酮硫解酶（phbA）和乙酰乙酰辅酶A还原酶（phbB）基因，并且还分别携带具有其自然启动子序列的phbC基因。此外，当在不添加葡萄糖的Luria-Bertani培养基中生长时，允许phbC基因在新的转录和翻译条件下表达的质粒pSYN与质粒pUMS相结合，在大肠杆菌中产生了相同量的PHB形成（占细胞干重70%的PHB）。
