高效除磷工程菌Pseudomonas putida GM6-PPK1的构建及其除磷能力研究

ppk1 基因编码的多聚磷酸盐激酶主要负责多聚磷酸盐的合成,其表达量的高低直接决定聚磷菌的聚 P 能力的强弱.Pseudomonas putida GM6 是从 EBPR 好氧池活性污泥中分离获得的一株具有聚 P 能力的菌株,该菌株含有两个编码多聚磷酸盐激酶的基因(ppk1 和 ppk2).通过 PCR 从高效聚磷菌株总 DNA 中扩增得到了 ppk1 及其启动子,并定向克隆到 pBBRMCS-5 载体上,构建了重组质粒 pMEPE-PPK,在辅助质粒 pRK2013 的帮助下,通过三亲接合将 pMEPE-PPK 转移到原始菌株 GM6 中,获得的工程菌 P. putida GM6-PPK1.GM6-PPK1 除 P 能力较原始菌株 GM6 和对照菌株 GM6-P5 提高了 54%,生长能力较原始菌株也有一定增强.通过模拟 EBPR 工艺,发现 GM6-PPK1 在厌氧/好氧交替的环境条件下强化表达不但提高了菌体好氧段的吸 P 能力,而且厌氧段 P 的释放和 PHA 的合成较之原始菌株也有显著增加.

Enhancement of phosphorus removal by overexpression of polyphosphate kinase 1 in Pseudomonas putida GM6

The Polyphosphate kinase responsible for poly-phosphate (polyP) synthesis is encoded by ppkl, the phosphate accumulating capacity of PAOs has a positive correlation with its ppkl gene expression level. Pseudomonas putida GM6 is an efficient phosphate accumulating strain isolated from EBPR system in the activated sludge aerobic pond, and ppk1 was cloned and characterized from GM6 previously. ppk1 gene and its promoter were amplified from the genomic DNA of GM6 by PCR. Recombinant plasmids pMEPE-PPK was constructed by ligating ppk1 gene and its promoter into broad host vector pBBRMCS-5. With the help of plasmid pRK2013, pMEPE-PPK was transferred into the strain GM6 to construct GM6-PPK1. The polyphosphate removal ability of GM6-PPK1 was increased by 54% compared with strain GM6 and its growth was also enhanced. In order to stimulate the SBR technique, the strains were grown under anaerobic and aerobic conditions. Results showed that GM6-ppk1 has stronger phosphate absorption ability in aerobic condition and it releases more phosphate and syntheses more PHA in anaerobic condition.