Metabolic Engineering of Aliphatic Glucosinolates in Hairy Root Cultures of Arabidopsis thaliana

Two cytochrome P450 proteins, CYP79F1 and CYP79F2, are involved in the biosynthesis of aliphatic glucosinolates (GSs) in brassicaceous species. In order to increase aliphatic GS levels in Arabidopsis thaliana hairy root cultures (HRCs) with the aim of biomanufacturing GSs, CYP79F1 and CYP79F2 from either Arabidopsis thaliana or Brassica rapa were first constitutively expressed in stably transformed Arabidopsis plants, and then HRC was induced by additional transformation with Agrobacterium rhizogenes. Gene expression levels and GS profiles were analyzed in HRC in order to validate the impact on aliphatic levels. To estimate the potency of gene transformation on GS content in HRC, GS levels were compared to levels in roots and leaves of transformed plants. According to the data obtained, short-chain aliphatic GSs in plants were raised in three generations after transformation, indicating that expression of transgenes was stable. After HRC induction, transcript levels of CYP79F1 and CYP79F2 rose and short-chain aliphatic GS levels increased by as much as 7-fold compared to the respective HRC wild-type control. However, when compared to levels present in leaves and roots of plants before HRC induction, aliphatic GS levels in HRC remained very low. In addition, expression of CYP79F1 and CYP79F2 also enhanced the content of indole GSs, mainly indol-3-ylmethyl GS and 4-hydroxy-indol-3-ylmethyl GS in HRC. The results presented provide basic information about GS biosynthesis in HRC of Arabidopsis thaliana compared to intact plants, which will help scientists to further optimize GS composition in HRC in the future.