聚羟基脂肪酸酯(PHA)是一类具有广泛应用前景的可降解生物塑料.因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视.目前的研究表明在积累中长链PHA的假单胞菌中,由phaG基因编码的(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶(PhaG)起关键作用,但目前为止对该蛋白还知之甚少.通过聚合酶链式反应(PCR)建立了一种快速、特异鉴定phaG基因的方法,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonas stutzeri 1317和Pseudomonas nitroreducens0802中分别克隆得到phaG基因,并在phaG基因突变株Pseudomonasputida PHAGN-21中表达成功.同时,还首次报道了从非假单胞菌菌株Burkholderia caryophylli AS 1.2741中鉴定得到phaG基因,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础.%Polyhydroxyalkanoates (PHA) were biodegradable thermoplastics. Due to their broad applications, direct biosynthesis of PHA from inexpensive substrates, such as carbohydrates, is actively pursued. It has been recently revealed that (R)-3-hydroxyacyl-ACP: CoA transacylase (PhaG) played an important role in this pathway. In this study, a polymerase chain reaction(PCR) protocol was developed for the rapid and specific identification of phaG gene from various bacteria. Using the PCR strategy, the complete open reading frames of two phaG genes from Pseudomonas stutzeri 1317 and Pseudomonas nitroreducens 0802were cloned from the genomic DNA and functionally expressed in Pseudomonas putida PHAGN-21. Furthermore, this strategy was successful applied in non-Pseudomonas strains, such as Burkholderia. These results suggest that PhaG-mediated pathway of medium-chain-length polyhydroxyalkanoates was widespread among bacteria.
展开▼
