倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究

Analysis gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase from fecal microbiome of Nycticebus pygmaeus

【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与Gen Bank中相应酶的序列一致性分别介于92%-100%和87%-100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有Lm PH(Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。

[Objective] The gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase were investigated from fecal microbiome of Nycticebus pygmaeus. [Methods] Degenerate primers were used to amplify phenol hydroxylase and catechol 1,2-dioxygenase gene fragments from metagenomic DNA. Phenol hydroxylase and catechol 1,2-dioxygenase gene clone libraries were constructed, and some of clones were sequenced separately. [Results] The BLAST analysis of phenol hydroxylase and catechol 1,2-dioxygenase sequences showed 92%-100% and 87%-100% identities to the known phenol hydroxylase and catechol 1,2-dioxygenase sequences in Gen Bank. Phylogenetic analysis showed that phenol hydroxylase sequences in gene clone libraries had high similarity with phenol hydroxylase sequences from Neisseria, Burkholderia, Alcaligenes, Acinetobacter. And catechol 1,2-dioxygenase sequences in gene clone libraries had high similarity with catechol 1,2-dioxygenase sequences from Acinetobacter. Sequence alignment showed two DEXRH motifs of Lm PH sequences were detected in phenol hydroxylase sequences, and the conserved cysteine was detected in catechol 1,2-dioxygenase sequences which was inhibited by Ag＋ and Hg2＋. [Conclusion] The phenol hydroxylase from fecal microbiome of Nycticebus pygmaeus was multicomponent phenol hydroxylase, and catechol that middle production of phenol degradation can be cleaved by catechol 1,2-dioxygenase through ortho-pathway.