Production of resveratrol in Flammulina velutipes by combinatorial expression of its biosynthetic genes

Flammulina velutipes is cultivated commercially throughout the world because of its rich nutrients and bioactive substances, especially the precursor for resveratrol biosynthesis, p-coumaric acid. In this paper, a bioconversion system was established for the production of resveratrol in F. velutipes. The expression vector pgfvs-4cl-vrs2 containing 4-coumarate: coenzyme A ligase gene (4cl) and resveratrol synthase gene (rs) from Vitis vinifera was constructed and transformed into F. velutipes. PCR, Southern blotting, and RT-PCR were used to identify positive transformants. Results indicated that the 4cl and rs genes have been successfully integrated into the genome of F. velutipes and could show normal transcription. High-Pressure Liquid Chromatography (HPLC) analysis indicated that the 4cl and rs genes were functionally expressed in three transformants, TF71, TF7H, and TF7E, which have an ability to convert p-coumaric acid into resveratrol. The contents of resveratrol for the transgenic strain TF71 were 2.77 μg/g, 1.60 μg/L, and 1.39 μg/g in the mycelia, fermentation liquid, and fruiting body (dry weight), respectively. The contents of the transgenic strain TF7H were 1.67 μg/g, 1.60 μg/L, and 1.01 μg/g, and those of the transgenic strain TF7E were 0.06 μg/g, 3.4 μg/L, and 1.13 μg/g, respectively. The present results indicated that establishing a metabolic pathway for resveratrol production in F. velutipes was feasible, thereby adding a new F. velutipes functional ingredient.