伯克霍尔德氏菌(Burkholderia sp.)2-萘酸单加氧酶基因(nmo)的克隆及表达

Cloning and Expression of a 2-Naphthoate Monooxygenase Gene (nmo) in Burkholderia sp. JT1500

通过酶切连接将Burkholderiasp.JT1 5 0 0的一段DNA片段 (4 8kb)亚克隆到表达载体pUC1 8上 ,得到重组子pEK1 2 3。测序后的pEK1 2 3重组子 4 8kb插入片段的序列已经登陆欧洲EMBL基因库 ,序列接受号为AJ5 663 3 3。对这一DNA片段的序列分析显示 ,此DNA片段含有 3个阅读框 ,且在这 3个阅读框 5′端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3 ,其阅读框长度为 1 1 5 8bp ,编码 3 86个氨基酸 ,与已报道的RalstoniaeutrophaHF3 9羟化酶 (单加氧酶 ,bec)氨基酸序列有 64%的同源性。pEK1 2 3对 2 萘酸代谢途径中 4个关键底物的转化实验结果显示 ,其基因产物仅对 2 萘酸发生加氧转化反应 ,而且 2 萘酸浓度有明显的降低 ,证实此基因是 2 萘酸单加氧酶基因 (nmo)。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中SDS PAGE结果表明 ,pXK3、pEK1 2 3两重组子中 2 萘酸单加氧酶表达量并没明显区别 ,但加氧酶酶活却存在显著的差别。推测在启动子后 ,单加氧酶阅读框前的两个阅读框的基因产物 ,对单加氧酶活有促进作用。

A 4 8kb DNA fragment from one blue colony of the pLARF1 gene library o f Burkholderia sp. JT1500 was subcloned to pUC18, designated as pEK123. The sequ ence of the inserted 4 8kb DNA of pEK123 was analyzed and submitted to EMBL nuc l eotide database, the accession# is AJ566333. The transformants of pEK123 could a lso become blue in LB agar and sequence analysis showed that three open reading frames and a putative promoter sequence were located in this inserted fragment. Then the 4 4kb insert fragment of pEK123 was double digested with XbaⅠ/ Kpn Ⅰ and EcoRⅠ/XbaⅠ respectively to construct plamsids pXK3 and pEX12 . The pXK3 contained only one 1158bp open reading frame (ORF) and pEX12 with other two ORFs. Unlike pEK123, the colonies of pEX12 did not show any blue color even incubated for 72h in LB agar, but the transformants of pXK3 did oxidize indole into indigo. The d educed 43kD protein of 1158bp ORF showed 64% homology of amino acid composition to Ralstonia eutropha HF39 hydroxylase(bec). Results of substrate transforma tion analysis showed that the transformants of pEK123 was able to catalyze the oxida tion of 2-naphthoate but not other key intermediates in 2-naphthoate metabolic p athway. These results confirmed that the product of 1158bp ORF is 2-naphthoate monooxygenase. Though the oxygenase activity of pEK123 is much higher than that of pXK3, SDS-P A GE analysis found no difference between the amount of the band of monooxygenase produced by pXK3 or pEK123, but one more band was found produced by pEK123. Acco rding to the difference of substrate analysis between pXK3 and pEK123, it is sup posed that the products of two open reading frames up stream of nmo gene had strong influence on the activity of the monooxygenase. Benzoate was oxidized by free-cell extracts of the transformants of pEK123 in the transformation experiment with different aromatic substrates. As the DNA seq uence and amino acid sequence of 2-naphthoate monoxygenase(nmo) did no show any homology with the DNA sequence and amino acid sequence of benzoate oxygenases re ported, the pathway of benzoate oxidation conducted by nmo is on the investigati on.