Liu S J, Steinbüchel A
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, D-48149, Münster, Germany.
Appl Environ Microbiol. 2000 Feb;66(2):739-43. doi: 10.1128/AEM.66.2.739-743.2000.
A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.
通过在大肠杆菌中同时表达丙酮丁醇梭菌的丁酸激酶（Buk）和磷酸转丁酰酶（Ptb）基因以及嗜硫小红卵菌的两个聚羟基脂肪酸酯合酶基因（phaE和phaC），构建了一条合成聚羟基脂肪酸酯（PHA）的新途径。将这四个基因克隆到pBR322的BamHI和EcoRI位点，得到的重组质粒pBPP1赋予大肠杆菌JM109所有三种酶的活性。当提供羟基脂肪酸作为碳源时，该重组菌株的细胞积累了PHA。从相应的羟基脂肪酸中分别获得了3-羟基丁酸（3HB）、4-羟基丁酸（4HB）或4-羟基戊酸（4HV）的均聚物。当以等量添加其中两种羟基脂肪酸时，也获得了这些羟基脂肪酸的各种共聚物：用3HB和4HB喂养的细胞积累了一种共聚物，其中3HB占88摩尔%，4HB占12摩尔%，占细胞干重的68.7%。用3HB和4HV喂养的细胞积累了一种共聚物，其中3HB占94摩尔%，4HV占6摩尔%，占细胞干重的64.0%。用3HB、4HB和4HV喂养的细胞积累了一种三元共聚物，其中3HB占85摩尔%，4HB占13摩尔%，4HV占2摩尔%，占细胞干重的68.4%。
