Matsusaki H, Manji S, Taguchi K, Kato M, Fukui T, Doi Y
Polymer Chemistry Laboratory and the RIKEN Group of Japan Science and Technology Corporation, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
J Bacteriol. 1998 Dec;180(24):6459-67. doi: 10.1128/JB.180.24.6459-6467.1998.
Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.
假单胞菌属61-3菌株能产生聚（3-羟基丁酸酯）[P(3HB)]均聚物和由4至12个碳原子的3HA单元组成的无规共聚物（聚（3-羟基丁酸酯-co-3-羟基链烷酸酯）[P(3HB-co-3HA)]）的混合物，其两种聚羟基链烷酸酯（PHA）生物合成基因座（phb和pha）在分子水平上被克隆并分析。在phb基因座中，发现了三个编码聚羟基丁酸酯（PHB）合酶（PhbCPs）、β-酮硫解酶（PhbAPs）和NADPH依赖性乙酰乙酰辅酶A还原酶（PhbBPs）的开放阅读框。基因组织显示有一个假定的启动子区域，随后是phbBPs-phbAPs-phbCPs。在phbBPs上游发现了phbRPs基因，它与转录激活因子的AraC/XylS家族成员具有显著相似性。发现phbRPs基因的转录方向与三个结构基因相反。将phbRPs克隆到假单胞菌属61-3菌株中相对高拷贝的载体中，提高了来自转录phb启动子-lacZ融合的β-半乳糖苷酶活性水平，并且还提高了该菌株合成的聚酯中3HB的比例，这表明PhbRPs是控制该细菌中phbBACPs转录的正调控蛋白。在pha基因座中，两个编码PHA合酶（PhaC1Ps和PhaC2Ps）的基因两侧是一个PHA解聚酶基因（phaZPs）以及两个相邻的开放阅读框（ORF1和phaDPs），基因顺序为ORF1、phaC1Ps、phaZPs、phaC2Ps和phaDPs。在恶臭假单胞菌和富养罗尔斯通氏菌的PHA阴性突变体中克隆片段的异源表达表明，假单胞菌属61-3菌株的PHB合酶和两种PHA合酶分别对短链长度以及短链和中链长度的3HA单元具有特异性。
