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

Enhancement of Phosphorus Removal by Overexpression of Polyphosphate Kinase 1 in Pseudomonas Putida GM6

作　　者：李波[1] 刘卫东[1] 刘娟[1] 赵晓丽[1] 高雅英[1] 曹慧[1] 崔中利[1] 

机构地区：[1]南京农业大学生命科学学院,农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《土壤》2009年第4期600-606,共7页Soils

基　　金：教育部新世纪优秀人才计划项目资助

摘　　要：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.putidaGM6-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。The Polyphosphate kinase responsible for poly-phosphate （polyP） synthesis is encoded by ppk1, the phosphate accumulating capacity of PAOs has a positive correlation with its ppk1 gene expression level. Pseudoraonas putida GM6 is an efficient phosphate accumulating strain isolated from EBPR system in the activated sludge aerobic pond, andppk1 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 ligatingppk1 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.

关 键 词：强化生物除磷 多聚磷酸盐 多聚磷酸盐激酶 基因工程菌 

分 类 号：S154.39[农业科学—土壤学]