钝齿棒杆菌N-乙酰谷氨酸激酶基因的克隆、序列分析及表达

以钝齿棒杆菌(Corynebacterium crenatum)野生株AS 1.542及产精氨酸突变株971.1的基因组为模板,用PCR方法扩增出N-乙酰谷氨酸激酶基因(argB)片段。核酸序列分析结果表明,该片段全长1505bp,包含一个ORF,推测此ORF区编码一条317个氨基酸的多肽,分子量为33.6kDa。C.crenatum野生株AS 1.542与突变株971.1的argB基因序列比较,发现只在结构区有一个核苷酸的差别但没有引起氨基酸变化。野生株AS 1.542argB基因的编码区核苷酸序列与C.glutamicumATCC 13032、Corynebacterium efficiensYS-314和Escherichia colik12的同源性分别是99.89%、76.62%和37.94%,而氨基酸同源性分别是100%、78.55%和25.25%。在C.crenatum argB基因上游存在启动子区域。经IPTG诱导该基因在棒杆菌中得到有效表达,野生株AS 1.542为宿主的重组子酶活明显提高。突变株971.1为宿主的重组菌酶活提高一倍,精氨酸积累提高约25%。

Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum

N-Acetylgutamate kinase(EC 2.7.2.8;NAGK) genes from wild-type Corynebacterium crenatum AS 1.542 and a L-arginine-producing mutant C.crenatum 971.1 were cloned and sequenced.Analysis of argB sequences revealed that only one ORF existed,which used ATG as the initiation codon and coded a peptide of 317 amino acids with a calculated molecular weight of 33.6kDa.Only one nucleotide difference was found in the structure gene and the difference did not cause a change of amino acid by comparision of the gene sequences between the wild type C.crenatum AS 1.542 and the mutant 971.1.The ORF sequence of argB from C.crenatum AS 1.542 showed homologies of 99.89%,76.62%,37.94% to those from Corynebacterium glutamicum ATCC 13032,Corynebacterium efficiens YS-314 and Escherichia coli k12.And the amino acid sequence deduced from ORF displayed homologies of 100%,78.55%,25.25% to those from microorganisms above,respectively.An internal promoter was found in the upstream of the argB gene from C.crenatum.The argB gene from C.crenatum AS 1.542 was expressed both in C.crenatum AS 1.542 and 971.1.The NAGK activity of transformed C.crenatum AS 1.542 was greatly increased by the induction of IPTG.The NAGK activity of transformed C.crenatum 971.1 was almost twice as much as that of C.crenatum 971.1 under the same induction.The amplification of the NAGK activity yielded 25% increase of L-arginine production in C.crenatum 971.1.