细菌Dietzia sp.DQ12-45-1b中两种长链烷烃羟化酶基因的鉴定

Identification of two alkane hydroxylases involved in long-chain n-alkane degradation by Dietzia sp.DQ12-45-1b

细菌Dietzia sp.DQ12-45-1b是一株高效石油降解菌,从中已经鉴定了编码膜整合烷烃羟化酶(Alk B)和细胞色素P450类CYP153烷烃羟化酶的基因alk W1和CYP153,该菌能产生不同的表面活性剂以降解不同碳链长度的烷烃,但在不同时期对不同碳源进行降解的机制仍不清楚.通过以二十八烷(C28)为单一碳源培养alk W1缺陷的突变株M 5-5,发现alk W1缺陷降低了菌株对C28的降解能力,但仍具有降解C28的能力.通过对DQ12-45-1b菌株全基因组信息分析,并运用实时荧光定量PCR(real-time PCR)技术,鉴定了两种长链烷烃羟化酶基因,其中G1的氨基酸序列与Acinetobacter sp.DSM17874 alm A的相似性是50%,G2的氨基酸序列与Geobacillus thermodenitrifi cans NG80-2 lad A的相似性是48%.同时,发现Dietzia sp.E1存在基因G1的同源基因(G1α)和G2的同源基因(G2α和G2β),其中基因G1α与G1的相似性是71%,基因G2α、G2β与G2的相似性分别是34%、87%.Real-time PCR分析表明,在Dietzia sp.DQ12-45-1b及突变株M 5-5中,基因G1和G2的转录水平均因C28的诱导而显著上调.上述对alm A和lad A同源基因的鉴定表明,Dietzia sp.DQ12-45-1b具有独特的烷烃降解多酶体系,是目前报道的烷烃羟化酶最多的细菌之一.

The aim of this study was to identify genes involved in long-chain alkane degradation in Dietzia sp.DQ12-45-1b.Functional genes were annotated by genome analysis.Induction of alkane hydroxylase genes by C28 n-alkane was analyzed by using quantitative real-time PCR in wild-type Dietzia sp.DQ12-45-1b and its alkW1 gene knockout mutant strain M 5-5.From the genome of Dietzia sp.DQ12-45-1b,two homologues,G1 and G2 genes were annotated,which showed 50%amino acid sequence similarity with AlmA from Acinetobacter sp.DSM17874,and 48%amino acid sequence similarity with LadA from Geobacillus thermodenitrificans NG80-2,respectively.In addition,G1 showed 71%amino acid sequence similarity with G1α,and G2 showed34%and 87%amino acid sequence similarities with G2α and G2β,respectively,which were annotated from Dietzia sp.E1 genome.In addition,the alkW1 gene knockout strain M 5-5 could grow with C28 n-alkane as the sole carbon source,indicating the presence of potential long-chain alkane hydroxylase gene（s） other than alkW1 in Diezia sp.DQ12-45-1b.Accordingly,induction of G1 and G2 genes was observed when Dietzia ap.DQ12-45-1b and alkWl knockout mutant strain M 5-5 grew with C28 n-alkane as sole carbon source.The results indicated that G1 and G2 genes are mostly responsible for the degradation of long-chain alkanes in Dietzia sp.DQ12-45-1b,which has unique multiple alkane hydroxylase systems.