Fukui T, Doi Y
Polymer Chemistry Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan.
J Bacteriol. 1997 Aug;179(15):4821-30. doi: 10.1128/jb.179.15.4821-4830.1997.
A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.
从豚鼠气单胞菌的基因组DNA中克隆并分析了一个5.0千碱基对的EcoRV-EcoRI限制性片段，豚鼠气单胞菌是一种能从链烷酸或油类生产（R）-3-羟基丁酸（3HB）和（R）-3-羟基己酸（3HHx）的共聚酯[聚（3HB-co-3HHx）]的细菌。该区域的核苷酸序列显示有一个1782碱基对的聚（3-羟基链烷酸酯）（PHA）合酶基因（phaC（Ac）[即来自豚鼠气单胞菌的phaC基因]），以及四个开放阅读框（ORF1、-3、-4和-5）和一个推定的启动子区域。克隆的片段不仅可以互补真养产碱菌和恶臭假单胞菌的PHA阴性突变体，还能赋予突变体宿主从辛酸或己酸合成聚（3HB-co-3HHx）的能力。此外，在真养产碱菌突变体中，ORF1和ORF3基因与phaC（Ac）共表达导致细胞中聚酯含量降低。表达ORF3的大肠杆菌显示出（R）-烯酰辅酶A（CoA）水合酶活性，这表明在豚鼠气单胞菌中，（R）-3-羟基酰基-CoA单体单元是通过烯酰-CoA的（R）-特异性水合作用提供的。仅表达phaC（Ac）的真养产碱菌突变体的转接合子在一步培养中能从辛酸有效地积累聚（3HB-co-3HHx），其含量高达细胞干重的96%。
