一步法生产1,5-戊二胺谷氨酸棒杆菌基因工程菌的构建

1,5-戊二胺是一种重要的化工原料,发酵法生产1,5-戊二胺是一条新颖且具有潜在竞争力的生产途径。以蜂房哈夫尼菌(Hafnia alvei)AS1.1009基因组为模板,通过PCR扩增,得到大小约为2.2kb的赖氨酸脱羧酶基因ldc。以大肠杆菌(Escherichia coli)/谷氨酸棒杆菌(Corynebacterium glutamicum)穿梭质粒pXMJl9为载体,将扩增得到的目的基因片段克隆至谷氨酸棒杆菌C.glutamicum TK260512,获得重组菌株C.glutamicum TK260512/pXMJl9-ldc.在摇瓶发酵水平上,通过IPTG诱导ldc基因的表达,并采用反相高效液相色谱方法测定了发酵液中1,5-戊二胺的含量,结果显示,经36h发酵,工程菌C.glutamicum TK260512/pXMJ19-ldc的1,5-戊二胺产量为0.96g/L。

Construction of Recombinant Corynebacterium glutamicum Producing 1,5-Pentanediamine by One Step Method

1,5-Pentanediamine is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1,5-Pentanediamine has been a novel and competitive way. An about 2.2 kb fragment of Hafnia alvei lysine decarboxylase gene encoding lysine decarboxylase (LDC) was proliferated by polymerase chain reaction by using chromosomal DNA of Hafnia alvei as the template. The obtained ldc fragment was inserted into E. coli/C. glutamicum shuttle vector pXMJ19 to construct an expression plasmid pXMJ19-ldc. It was then introduced into C. glutamicum TK260512 via electrotransformation, and a recombinant C. glutamicum TK260512/pXMJl9-ldc was obtained. The 1,5-Pentanediamine in the broth was detected by HPLC when the culture in the shake flask was indueed with 0.5 mmol/L IPTG. The results showed that after 36 h cultivation,the recombinant stain could produce 0.96 g/L 1,5-Pentanediamine.