Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g(-1) fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host.