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Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus

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Abstract

Various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase were added to a Catharanthus roseus cell culture medium to investigate the regulatory effects on tabersonine, vindoline and vinblastine biosynthesis. Hydrogen peroxide was found to be the most effective agent for enhancing the biosynthesis of tabersonine. By adding 20 μg/L hydrogen peroxide, the tabersonine concentration reached 9.02 mg/g dry weight (DW) after culturing cell suspensions for 7 days. With the addition of 30 μg/L acetyl CoA, the most vindoline (final cell content of 0.33 mg/g DW) was produced. By effective inhibition of lochnericine biosynthesis with the addition of 0.5 μmol/L benzotriazole, the cell content of vindoline was increased to 0.42 mg/g DW. An orthogonal experiment consisting of multiple regulation factors was carried out to optimize vinblastine biosynthesis. It was shown that optimal vinblastine biosynthesis was achieved by addition of 5 mg/L acetyl CoA, 20 μg/L hydrogen peroxide, 0.5 μmol/L benzotriazole, 100 mg/L tryptophan, 100 mg/L loganin and 30 mg/L cerium chloride. Under these conditions, the cell content of vinblastine reached 0.81 mg/g DW. Simultaneous changes in cell content and enzyme activities of Cytochrome P-450 monooxygenases, Deacetylvindoline-O-acetyltransferase and Peroxidase enzyme indicated that these enzymes were closely linked to vinblastine biosynthesis.

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Abbreviations

T16H:

Tabersonine-16-hydroxylase

DAT:

Deacetylvindoline-O-acetyltransferase

STR:

Strictosidine synthase

LCO:

Lochnericine cyclooxygenase

POD:

Peroxidase enzyme

P-450:

Cytochrome P-450 monooxygenases

G10H:

Geraniol-10-hydroxylase

D4H:

Deacetoxyvindoline 4-hydroxylase

SOD:

Superoxide dismutase

CAT:

Catalase

TDC:

Tryptophan decarboxylase

DTT:

Dithiothreitol

HA:

Hydroxyphenylpyruvic acid

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Author notes

  1. Mei-Zhen Gong

    Present address: Chemistry and Life Science College, China Three Gorges University, Hubei, 443002, China

Authors and Affiliations

  1. Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

    Zhi-Gang Guo, Yun Liu, Mei-Zhen Gong, Wen Chen & Wen-Yuan Li

Authors
  1. Zhi-Gang Guo
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  2. Yun Liu
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  3. Mei-Zhen Gong
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  4. Wen Chen
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Corresponding author

Correspondence to Zhi-Gang Guo.

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11240_2012_213_MOESM1_ESM.tif

LC image of Catharanthus roseus cells’ extraction; The retention times of catharanthine, vindoline, vinblastine, and tabersonine were 4.07, 10.36, 11.34 and 14.76 min, respectively. Below was the MS of the extraction when retention time is 4.06 min. We indicate that it should be catharanthine (TIFF 1053 kb)

11240_2012_213_MOESM2_ESM.tif

MS of the extraction when retention time was 7.66 min, we indicate that it was catharanthine (molecular weight, 336.43), below was the MS of the extraction when retention time was 10.36 min, we indicate that it was vindoline (molecular weight, 456.54) (TIFF 1068 kb)

11240_2012_213_MOESM3_ESM.tif

MS of the extraction when retention time was 11.34 min, we indicate that it was vinblastine (molecular weight, 810.9); below is the MS of the extraction when retention time was 14.76 min, we indicated that it was tabersonine (molecular weight, 336.43) (TIFF 909 kb)

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Guo, ZG., Liu, Y., Gong, MZ. et al. Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus . Plant Cell Tiss Organ Cult 112, 43–54 (2013). https://doi.org/10.1007/s11240-012-0213-y

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  • DOI: https://doi.org/10.1007/s11240-012-0213-y

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