Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants-Ministry of Education, College of Forestry, Hainan University, Haikou 570228, China.
Int J Mol Sci. 2022 Jun 17;23(12):6780. doi: 10.3390/ijms23126780.
, an endemic medicinal herb in Hainan Island, is rich in bibenzyls. However, the key rate-limited enzyme involved in bibenzyl biosynthesis has yet to be identified in . In this study, to explore whether there is a significant difference between the tissues, the total contents of bibenzyls were determined in roots, pseudobulbs, and leaves. The results indicated that roots had higher bibenzyl content than pseudobulbs and leaves. Subsequently, transcriptomic sequencings were conducted to excavate the genes encoding type III polyketide synthase (PKS). A total of six () genes were identified according to gene function annotation. Phylogenetic analysis classified the type III genes into three groups. Importantly, the c93636.graph_c0 was clustered into bibenzyl synthase (BBS) group, named as BBS (DsBBS). The expression analysis by FPKM and RT-qPCR indicated that showed the highest expression levels in roots, displaying a positive correlation with bibenzyl contents in different tissues. Thus, the recombinant DsBBS-HisTag protein was constructed and expressed to study its catalytic activity. The molecular weight of the recombinant protein was verified to be approximately 45 kDa. Enzyme activity analysis indicated that the recombinant DsBBS-HisTag protein could use 4-coumaryol-CoA and malonyl-CoA as substrates for resveratrol production in vitro. The Vmax of the recombinant protein for the resveratrol production was 0.88 ± 0.07 pmol s mg. These results improve our understanding with respect to the process of bibenzyl biosynthesis in .
海南粗榧,一种海南特有的药用植物,富含联苄类化合物。然而,在海南粗榧中,参与联苄生物合成的关键限速酶尚未被鉴定。在这项研究中,为了探索 组织之间是否存在显著差异,测定了根、假鳞茎和叶中联苄的总含量。结果表明,根中的联苄含量高于假鳞茎和叶。随后,进行了转录组测序以挖掘编码 III 型聚酮合酶(PKS)的基因。根据基因功能注释,共鉴定出 6 个 () 基因。系统发育分析将 III 型 基因分为三组。重要的是,c93636.graph_c0 被聚类到联苄合酶(BBS)组,命名为 BBS(DsBBS)。通过 FPKM 和 RT-qPCR 的表达分析表明, 在根中表达水平最高,与不同组织中联苄含量呈正相关。因此,构建并表达了重组 DsBBS-HisTag 蛋白以研究其催化活性。重组蛋白的分子量约为 45 kDa。酶活性分析表明,重组 DsBBS-HisTag 蛋白可以使用 4-香豆酰辅酶 A 和丙二酰辅酶 A 作为底物在体外合成白藜芦醇。该重组蛋白生产白藜芦醇的 Vmax 为 0.88 ± 0.07 pmol s mg。这些结果提高了我们对海南粗榧中联苄生物合成过程的理解。