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Further Optimization of Heterologous Glucoraphanin Biosynthesis Through Co‐Expression Analysis
Author(s) -
Pignolet Roxanna,
Endelman Benjamin,
Barnum Collin,
Shih Patrick M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04133
Subject(s) - glucoraphanin , heterologous , heterologous expression , gene , chemistry , biosynthesis , food science , biochemistry , biology , botany , recombinant dna , brassica , glucosinolate
It is estimated that 38.4% of adults in the United States will be diagnosed with cancer (National Cancer Institute). With this high rate of incidence, the exploration of new options for cancer prevention and treatment is of high importance. Plant secondary metabolites are strong candidates for study due to their potential to be used in anti‐cancer dietary supplements. Isothiocyanates are products of the glucoraphanin biosynthesis pathway that have been demonstrated to display potent anti‐cancer and anti‐inflammatory properties. Previous studies have identified the genes in the core glucoraphanin biosynthesis pathway (CGBP); however, heterologous expression of the CGBP in tobacco has produced glucoraphanin at only 0.35 μmol g‐1 dry weight (Mikkelsen et al. 2010). In order to increase our titer, we used co‐expression analysis to identify a total of forty‐two gene candidates for optimizing the CGBP. We tested these candidates for their effect on glucoraphanin biosynthesis by transiently expressing the CGBP with candidate genes in tobacco. LC‐MS analysis of the transformed leaf tissue for glucoraphanin content revealed that six of the forty‐two genes increased glucoraphanin production. We also found several genes that produced significantly less glucoraphanin per gram dry weight when combined with the CGBP than the CGBP alone. The increases in heterologous glucoraphanin production implicate the six genes as important factors in the glucoraphanin biosynthetic pathway with the potential to raise concentrations closer to recommended supplement quantities. Support or Funding Information This project is funded by NIH Grant R00AT009573