Ueda S, Yabutani T, Maehara A, Yamane T
Department of Bioproductive Sciences, Faculty of Agriculture, Utsunomiya University, Japan.
J Bacteriol. 1996 Feb;178(3):774-9. doi: 10.1128/jb.178.3.774-779.1996.
A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaCPd. A plasmid vector carrying the phaCPd'-'lacZ fusion gene downstream of the promoter-like sequence expressed beta-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.
反硝化副球菌DNA的一个3.6千碱基对的EcoRI-SalI片段与携带嗜碱产碱菌聚（3-羟基链烷酸酯）（PHA）合酶基因（phaC）的DNA探针杂交。该区域的核苷酸序列分析显示存在一个1872碱基对的开放阅读框（ORF），它对应于一个分子量为69537的多肽。在ORF的上游，发现了一个类似启动子的序列。携带lacZ与ORF之间融合基因的大肠杆菌在存在嗜碱产碱菌的β-酮硫解酶和乙酰乙酰辅酶A还原酶基因的情况下，积累的聚（3-羟基丁酸酯）水平高达细胞干重的20%（重量）。该ORF被命名为phaCPd。一个携带phaCPd'-'lacZ融合基因且位于类似启动子序列下游的质粒载体在反硝化副球菌中表达β-半乳糖苷酶活性。当一个携带ORF以及类似启动子序列的多拷贝和广宿主范围载体被导入反硝化副球菌时，与仅携带载体序列的细胞相比，细胞中的PHA含量增加了两倍。
