Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

Full-length transcriptome, proteomics and metabolite analysis reveal candidate genes involved triterpenoid saponin biosynthesis in Dipsacus asperoides.

Dipsacus asperoides is a traditional medicinal herb widely used in inflammation and fracture in Asia. Triterpenoid saponins from D. asperoides are the main composition with pharmacological activity. However, the biosynthesis pathway of triterpenoid saponins has not been completely resolved in D. asperoides. Here, the types and contents of triterpenoid saponins were discovered with different distributions in five tissues (root, leaf, flower, stem, and fibrous root tissue) from D. asperoides by UPLC-Q-TOF-MS analysis. The discrepancy between five tissues in D. asperoides at the transcriptional level was studied by combining single-molecule real-time sequencing and next- generation sequencing. Meanwhile, key genes involved in the biosynthesis of saponin were further verified by proteomics. In MEP and MVA pathways, 48 differentially expressed genes were identified through co-expression analysis of transcriptome and saponin contents, including two isopentenyl pyrophosphate isomerase and two 2,3-oxidosqualene ß-amyrin cyclase, etc. In the analysis of WGCNA, 6 cytochrome P450s and 24 UDP- glycosyltransferases related to the biosynthesis of triterpenoid saponins were discovered with high transcriptome expression. This study will provide profound insights to demonstrate essential genes in the biosynthesis pathway of saponins in D. asperoides and support for the biosynthetic of natural active ingredients in the future.

[Cloning and bioinformatics analysis of ß-amyrin synthase in Dipsacus asper].

Dipsaci Radix is one of the commonly used Chinese medicinal materials in China, with a long history. It has the medicinal activities of nourishing liver and kidney, recovering from broken sinews, and treating bone fracture. Triterpenoid saponins are the main functional ingredients of Dipsacus asper. ß-Amyrin synthases(ß-AS) as a superfamily of oxidosqualene cyclases(OSCs) can catalyze the construction of the skeleton structure of oleanane-type triterpenoid saponins. There are only a few studies about the ß-AS in D. asper, and the catalytic mechanism of this enzyme remains to be explored. To enrich the information of ß-AS, according to the transcriptome sequencing results, we cloned DaWß-AS gene from D. asper into a specific vector for heterologous expression in Escherichia coli. In the meantime, real-time PCR was performed to analyze the relative expression of DaWß-AS in four different tissues of D. asper. The results of RT-qPCR showed DaWß-AS had the highest expression level in leaves. Bioinformatics results indicated that DaWß-AS had a conserved domain of PLN03012 superfamily, belonging to the cl31551 superfamily. There was no transmembrane domain or signal peptide in DaWß-AS. This study provides a scientific basis for revealing the biological pathways of triterpenoid saponins in D. asper, which will facilitate the biosynthesis of the associated saponins and afford reference for the cultivation and development of high-quality resources of D. asper.