罗汉果2个氧化鲨烯环化酶基因克隆及功能表征

Cloning and functional characterization of two oxidosqualene cyclase genes from Siraitia grosvenorii

氧化角鲨烯环化酶(oxidosqualene cyclase, OSC)属于多基因家族,可以将常见的前体2,3-氧化角鲨烯转化为各种类型的三萜骨架。该研究根据罗汉果转录组数据分析设计引物,克隆得到2条罗汉果OSC基因SgAS1(GenBank No. QDO67189.1)和SgAS2 (GenBank No. QDO67190.1)。SgAS1的开放阅读框(open reading frame, ORF)为2 262 bp,编码754个氨基酸,SgAS2的ORF为2 289 bp,编码762个氨基酸。实时荧光定量PCR结果显示2条SgOSCs基因在茎、叶和果实的不同时期表现出不同的表达模式。系统发育分析表明,SgAS1和SgAS2都与β-香树脂醇合酶聚为一支,但进一步利用酵母异源表达进行功能表征发现SgAS1没有活性,SgAS2能产生β-香树脂醇作为唯一产物。多重序列比对表明,SgAS2具有与β-香树脂醇生物合成相关的保守MWCYCR序列,而SgAS1有一个不寻常的LFCYTR序列,对此作者进行了定点突变分析该序列并发现色氨酸残基(W)为影响SgOSCs功能的关键氨基酸残基。此外,作者将单功能β-amyrin合酶SgAS2导入课题组前期改造的底盘菌株GH1中,将β-amyrin的产量提高至44.05 mg·L-1。该研究首次报道了来自罗汉果中的单功能β-amyrin合酶SgAS2并对其进行了定点突变和合成生物学研究,为三萜化合物的定向生物合成提供了宝贵的资源。

Oxidosqualene cyclases(OSCs), belonging to a multigene family, can convert a common precursor 2,3-oxidosqualene into various types of triterpene skeletons. In this study, primers were designed according to the analysis of Siraitia grosvenorii transcriptome data, and two OSC genes SgAS1(GenBank No. QDO67189.1) and SgAS2(GenBank No. QDO67190.1) were cloned. The open reading frame(ORF) of SgAS1 was 2 262 bp, encoding 754 amino acids, and the ORF of SgAS2 was 2 289 bp, encoding 762 amino acids. Real-time quantitative PCR results demonstrated that the two SgOSCs genes showed different expression patterns in stems, leaves, and different stages of fruits. Phylogenetic analysis showed that both SgAS1 and SgAS2 were clustered with β-amyrin synthases into a branch, but further functional characterization using yeast heterologous expression found that SgAS1 was inactive and SgAS2 could produce β-amyrin as the sole product. Multiple sequence alignments revealed that SgAS2 had a conserved MWCYCR sequence related to β-amyrin biosynthesis, while SgAS1 had an unusual LFCYTR sequence, for which the authors performed site-directed mutagenesis analysis of this sequence and found that tryptophan residue(W) was the key amino acid residue that affected the function of SgOSCs. In addition, the authors transformed the monofunctional β-amyrin synthase SgAS2 into the chassis strain GH1, which was previously modified by the research group, and increased the yield of β-amyrin to 44.05 mg·L-1. This study first reported the monofunctional β-amyrin synthase SgAS2 from S. grosvenorii and conducted site-directed mutagenesis and synthetic biology investigation on it, providing a valuable resource for the directed biosynthesis of triterpenoids.