Consumption of cruciferous vegetables is associated with reduced risk of developing cancer, a phenomenon attributed to glucosinolates, which are characteristic of these vegetables. We report production of the bioactive benzylglucosinolate in the noncruciferous plant Nicotiana benthamiana through metabolic engineering. The study includes identification of gamma-glutamyl peptidase 1 (GGP1), which substantially increased glucosinolate production by metabolizing an accumulating glutathione conjugate, an activity not previously described for glucosinolate biosynthesis or for proteins containing glutamine amidotransferase domains.

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the g-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant g-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.

The red tide ciliate Mesodinium rubrum (= Myrionecta rubra) is known to contain symbionts of cryptophyte origin. Molecular data have shown that the symbiont is closely related or similar to free-living species of the Teleaulax/Plagioselmis/Geminigera clade. This suggests that the symbiont of M. rubrum is either a temporary symbiont or a quite recently established symbiont. Here, we present data from a number of experiments in which we offered M. rubrum a phototrophic dinoflagellate and 8 different cryptophyte species belonging to 5 different clades. Mesodinium rubrum was only able to grow when fed the 2 cryptophyte species belonging to the genus Teleaulax, T. acuta and T. amphioxeia. Using the nucleomorph large subunit rDNA gene as marker, we were able to discriminate the 2 Teleaulax species, allowing monitoring of the exchange of the symbionts in M. rubrum. Over a period of 35 d, M. rubrum was able to exchange its symbionts from T. amphioxeia symbionts to T. acuta symbionts. This research suggests that M. rubrum can only utilize prey within the Teleaulax/Plagioselmis/Geminigera clade for sustained high growth rates and provides the first time-frame of endosymbiont replacement by M. rubrum.

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

BackgroundMetabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.ResultsTo optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2) alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold.ConclusionAdjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in heterologous hosts. Our study emphasizes the importance of considering co-substrates and their biological nature in metabolic engineering projects.