Dendrobium sinense, 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 D. sinense. In this study, to explore whether there is a significant difference between the D. sinense 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 D. sinense PKS (DsPKS) genes were identified according to gene function annotation. Phylogenetic analysis classified the type III DsPKS genes into three groups. Importantly, the c93636.graph_c0 was clustered into bibenzyl synthase (BBS) group, named as D. sinense BBS (DsBBS). The expression analysis by FPKM and RT-qPCR indicated that DsBBS 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⁻¹ mg⁻¹. These results improve our understanding with respect to the process of bibenzyl biosynthesis in D. sinense.

Dendrobium sinense, a native orchid species of Hainan Island, is cultivated for its ornamental flowers. Recently, this species has gained significant attention due to its medicinal value. This study focuses on the identification of type III polyketide synthase (PKS), which catalyzes the formation of crucial intermediates in secondary metabolites. Through analysis of previous transcriptome data, a total of ten type III DsPKS genes were identified. Phylogenetic analysis categorized the type III PKS proteins into CHS, BBS, and PKS groups. Interestingly, the DsCHS3 gene exhibited alternative first exons, resulting in two splice variants, namely DsCHS3-1 and DsCHS3-2. Full-length cDNA sequencing revealed that DsCHS3-1 was the more prevalent splice variant. Prokaryotic expression and purification of DsCHS3-1 and DsCHS3-2 proteins were successfully achieved. Enzyme activity analysis demonstrated significantly higher catalytic activity in DsCHS3-2 compared to DsCHS3-1, particularly in the conversion of p-coumaryol-CoA and malonyl-CoA to naringin chalcone. Functional complementation assays in Arabidopsis mutants confirmed the higher catalytic activity of DsCHS3-2, as it restored flavonoid biosynthesis to a greater extent compared to DsCHS3-1. Overall, these findings offer valuable insights into the alternative splicing patterns and functional divergence of DsCHS3 genes in D. sinense.