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

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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.

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Acknowledgments

We thank Prof. Gerhard Sandmann and Dr. Norihiko Misawa for providing plasmid of pACCAR25ΔcrtX. This research project was financially supported by the Public Science and Technology Research Funds Projects of Ocean (201305022), Xiamen Southern Oceanographic Center funds (No.14GZP021NF21, No. 14CZP028HJ02), and Special Funds for Technical Development of Scientific Research Institutes (2012EG149243).

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    Authors and Affiliations

    1. Engineering Research Center of Marine Biological Resource Comprehensive Utilization, Third Institute of Oceanography, State Oceanic Administration, 184 Daxue Road, Xiamen, Fujian Province, 361005, People’s Republic of China

      Sulin Lou, Liuying Wang, Lijuan He, Zhaokai Wang & Xiangzhi Lin

    2. Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao City, Shandong Province, People’s Republic of China

      Guangce Wang

    3. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao City, Shandong Province, People’s Republic of China

      Guangce Wang

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    1. Sulin Lou
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    2. Liuying Wang
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    3. Lijuan He
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    Correspondence to Xiangzhi Lin.

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    Liuying Wang and Lijuan He contributed equally to this work.

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    Fig. S1

    Comparison of the predicted amino acid sequences of ZCD1 and CsZCD. Different amino acids are indicated with gray backgrounds. (DOC 26 kb)

    Fig. S2

    Enzymatic assays of ZCD1 and CCD2 in E.coli. (a) ZCD1 and CCD2 expressed in E. coli harboring the plasmid pACCAR25ΔcrtX, which accumulates zeaxanthin. Plasmid pACCAR25ΔcrtX was co-transformed into E. coli DH5α with and without empty vector pUC19, pUC19-ZCD1 or pUC19-CCD2, respectively. Induced for 48 h at 28 °C with 0.5 mM IPTG, the bacterium solution of ZCD1 and CCD2-expressing were discolored, compared with the ones of expressing pACCAR25ΔcrtX or empty vector pUC19. (b) HPLC analysis of acetone extracts of E. coli. (GIF 44 kb)

    High resolution image (TIFF 1949 kb)

    Fig. S3

    Screening of positive clones of Chlorella vulgaris from Agrobacterium tumefaciens-mediated transformation (a) C. vulgaris protoplasts were coated on the hygromycin selective plates. (b) and (c) C. vulgaris protoplasts after cocultivation with A. tumefaciens strains of EHA105 and LBA4404 that harbored the expression vector pCAMBIA1302-crtRB-ZCD1 were coated on the hygromycin selective plates. (GIF 133 kb)

    High resolution image (TIFF 7472 kb)

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    Lou, S., Wang, L., He, L. et al. Production of crocetin in transgenic Chlorella vulgaris expressing genes crtRB and ZCD1 . J Appl Phycol 28, 1657–1665 (2016). https://doi.org/10.1007/s10811-015-0730-2

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