Production of crocetin in transgenic Chlorella vulgaris expressing genes crtRB and ZCD1

Abstract

Crocetin is one of the major active ingredients of the rare medicinal herb saffron (Crocus sativus L.), also known as crocus. Crocetin has a wide range of effects including antitumor, anticancer, antioxidant, and antiatherosclerotic activity. Crocetin is currently only obtained via extraction from crocus stigmas, with limited yields. This study developed a new method of production of crocetin from genetically engineered Chlorella vulgaris, a unicellular, fast-growing, and heterotrophically cultured green microalga. A plant expression vector carrying the genes crtRB and ZCD1, which encode enzymes controlling critical steps in crocetin biosynthesis, was introduced into C. vulgaris using the Agrobacterium tumefaciens-mediated transformation method. After hygromycin screening, resistant C. vulgaris strains were achieved. Polymerase chain reaction amplification confirmed that the crtRB and ZCD1 were successfully integrated into the C. vulgaris genome. High-performance liquid chromatography analysis of methanol extraction showed that the transgenic C. vulgaris was able to produce crocetin, while wild-type C. vulgaris was not. These results strongly suggest that introducing crtRB and ZCD1 into C. vulgaris can successfully produce crocetin. This is a new method of crocetin production in C. vulgaris to compensate for the shortage of crocetin.