基因工程菌Pseudomonas sp. B4的构建及其产邻苯二酚发酵条件的初步研究  

The Construction of Recombinant Strain Pseudomonas sp. B4 and the Optimization of Fermentation Conditions

作　　者：唐咸来[1,2] 熊建文[1,3,4] 梁璇[1,3,4] 申佩弘[1,3,4] 武波[1,3,4] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西壮族自治区科学技术厅,南宁530005 [3]微生物及植物遗传工程教育部重点实验室,南宁530005 [4]广西亚热带生物资源保护利用重点实验室,南宁530005

出　　处：《中国生物工程杂志》2008年第10期79-83,共5页China Biotechnology

基　　金：广西科技攻关项目(桂科攻0537012);国家"863"计划(2003AA214040)资助项目

摘　　要：利用PCR技术以Pseudomonas sp.B3-1基因组DNA为模板,扩增出2.9kb编码苯甲酸双加氧酶基因簇benABC。将该基因簇连接于pLAFRJ载体,电转化至E.coliDH5α,再通过三亲本结合法导入野生菌株Pseudomonas sp.B3-1中,得到了一株邻苯二酚产量提高的基因工程菌,命名为Pseudomonas sp.B4,在相同发酵条件下,工程菌Pseudomonas sp.B4的邻苯二酚产量达到0.5mg/ml,比野生菌株Pseudomonas sp.B3-1提高了17.5%。对工程菌Pseudomonas sp.B4进行发酵条件优化表明,当苯甲酸钠浓度为6.0g/L,聚蛋白胨浓度为2.5g/L,温度为32℃以及pH值为6.0时,工程菌在200r/min旋转摇床发酵36小时后,邻苯二酚产量达到0.7mg/ml,比野生菌株Pseudomonas sp.B3-1提高了75%。The benzoate dioxygenase gene （benABC） cluster was PCR amplified from Pseudomonas sp. B3- 1 genomic DNA and cloned into the plasmid pLAFRJ to construct an expression plasmid. The recombinant plasmid was transformed into E. coli DH5α by electroporation and then transferred into Pseudomonas sp. B3-1 by triconjugation using pRK2073 as the helper plasmid. As a result, one recombinant strain Pseudomonas sp. B4 produce catechol reached 0. 5 mg/ml were obtained, which was 17. 5% higher than that of the strain Pseudomonas sp. B3-1. Subsequently, optimization experiments were carried out for the strain Pseudomonas sp. B4. The results showed that the optimal condictions for the strain to produce catechol were ： Sodium benzoate 6.0 g/L, polypepton 2.5 g/L, pH6.0, incubated at 32℃ and shaken at 200r/min for 36 h. The catechol yield reached 0. 7 mg/ml, which was 75% higher than that of the wild strain.

关 键 词：假单胞菌 苯甲酸钠 苯甲酸双加氧酶基因簇 邻苯二酚 

分 类 号：Q819[生物学—生物工程]