Klebisella oxytoca HD79乙酸激酶部分基因(ack)的克隆与序列分析

Sequence Analysis and Clone of the Partial Acetate Kinase Gene(ack) from Klebisella oxytoca HD79

乙酸是2,3-丁二醇典型代谢途径中的主要副产物之一,它的大量生成能够造成环境p H值的改变并且抑制菌体的生长,并对2,3-丁二醇的代谢产生影响。乙酸激酶是乙酸生成的关键酶,敲除其相关基因,理论上可以减少乃至消除乙酸的产生,从而提高2,3-丁二醇的产量和得率。本研究根据Klebisella oxytoca KCTC1686和Klebisella oxytoca HD79乙酸激酶基因ack序列的相似性设计引物,以产酸克雷伯氏菌基因组DNA和质粒载体p T-ack为模板,利用PCR克隆得到了乙酸激酶部分基因ack,长度为856bp,无碱基缺失。ORF分析发现该部分基因没有完整的开放阅读框,但是全部位于Klebisella oxytoca KCTC1686 ack基因的开放阅读框内。结果表明该片段为Klebisella oxytoca HD79乙酸激酶部分基因序列,为后续构建产酸克雷伯氏菌乙酸激酶突变株奠定了基础。

Acetic acid is one of the major metabolic by- products in 2,3- butanediol classical metabolicpathway. Production of 2,3-butanediol can be affected by synthesis of numerous acetic acid which will lead tochanging of environmental p H and inhibition of cell growth. Acetate kinase is a key enzyme in acetic acidmetabolic pathway, knock-out of encoding genes of acetate kinase will theoretically decrease and even removethe production of acetic acid, thus the yield and productivity of 2,3-butanediol will be enhanced. In this paper,856 bp of partial kinase gene ack without base deletion was cloned taking genomic DNA of Klebsiella oxytoca and plasmid p T- ack as templates with primers designed according to similarity of ack sequences from Klebisella oxytoca KCTC1686. The ack gene was located in open reading frame of ack gene of Klebisella oxytoca KCTC1686 though integrated ORF was not found in it by ORF analysis. The result demonstrated thatthe ack gene was partially acetate kinase gene from Klebisella oxytoca HD79 which laid the foundation for further construction of acetate kinase mutant of Klebisella oxytoca HD79 and provided reference for otherrelative researches.