SOE-PCR法构建新型PHA嵌合酶研究

Novel PHA Chimeric Enzyme Constructed by SOE-PCR

聚羟基脂肪酸酯(Polyhydroxyalkanonate,PHA)合酶是PHA合成过程中的关键酶,其活性和底物特异性决定PHA含量和单体组成.构建嵌合酶是改变PHA单体组成的一种有效方式.利用重叠延伸PCR(gene splicing by overlap extension,SOE-PCR)法,以富氧产碱菌和嗜水气单胞菌PHA合酶基因为亲本构建新的PHA嵌合酶.结果表明,带有新型嵌合酶基因的重组菌株AHRE可利用葡萄糖酸钠和果糖产生3HB.利用辛酸钠、月桂酸、油酸、辛酸钠和葡萄糖酸钠混合碳源,除3HB和3HHx外,还产生了新的单体组分3HO.辛酸钠和葡萄糖酸钠混合碳源发酵,PHA含量最高,达57.54%.辛酸钠为单一碳源时,3HO单体组成含量最高,占PHA总量1.932%,证明嵌合酶构建成功.这为今后利用基因工程技术改变菌株PHA单体组成提供了有效方法,为获得具有更好材料学特性的PHA奠定了工作基础.

PHA synthase plays a central role in PHA biosynthesis.The activity and substrate specificity of PHA synthase determine the content and composition of PHA.The construction of chimeric enzyme is an effective way to change the composition of PHA monomer.In this study,PHA synthase genes from Ralstonia Eutropha and Aeromonas hydrophila used as parents,a new chimeric PHA synthase was constructed by SOE-PCR.The results showed that the recombinant strain harboring chimeric synthase gene only produced 3HB when fructose and gluconate as carbon source.In addition to 3HB and 3HHx,a new component 3HO was produced when sodium caprylate,lauric acid,oleic acid,gluconate and sodium caprylate as mixed carbon source.The highest PHA was obtained when fermented with a mixture of gluconate and sodium octanoate,up to 57.54%.When sodium acid was used as a single carbon source,the composition content of 3HO monomer was the highest,accounting for 1.932%of the total PHA.It was shown that the chimeric enzyme was successfully constructed.This provides an effective method for using genetic engineering techniques to change the composition of PHA monomer of strains in the future,and lays a working foundation for obtaining PHA with better material properties.