中长链聚羟基脂肪酸酯(mcl PHA)在嗜水气单胞菌I型PHA合酶缺失突变株中的合成(英文)

Synthesis of Medium-chain-length Polyhydroxyalkanoate (mcl PHA) in Type I PHA Synthase Negative Mutant of Aeromonas hydrophila

拥有Ⅰ型聚羟基脂肪酸酯(PHA)合酶基因的嗜水气单胞菌CGMCC0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因(Cm)插入到该基因中,获得带有I型PHA合酶断裂基因(phaC::Cm)的自杀质粒pFH10。自杀质粒pFH10通过接合作用转入嗜水气单胞菌CGMCC0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase gene (phaC) only accumulate copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, from lauric acid as sole carbon source but not from glucose. The gene encoding type I PHA synthase was interrupted by insertion of a chloramphenicol resistance gene (Cm). Conjugation of suicide plasmid pFH10 transformed A. hydrophila CGMCC 0911 into a recombinant organism with the disrupted type I PHA synthase gene (phaC::Cm), through an in vivo homologous recombination process, type I phaC of A. hydrophila genome was replaced by the disrupted phaC, and Cm gene was integrated into the genome of A. hydrophila, resulting in type I phaC-negative mutant, which was proved by DNA sequencing. Results of GC analysis showed that this mutant could not accumulate PHBHHx again but accumulate medium-chain-length (mcl) PHA from lauric acid or glucose as carbon source, clearly indicating the existence of another type Ⅱ PHA synthase in the wild type A. hydrophila. It will play its function and accumulate mcl PHA only when type I PHA synthase was inactivated.