METDEF · Metabolic engineering of triterpenoid pathways involved in plant defense in Arabidopsis and rice

Metabolic engineering of triterpenoid pathways involved in plant defense in Arabidopsis and rice

The EU funded research project was focused on the engineering of the triterpene metabolic pathway into Arabidopsis and rice through synthetic biology. Triterpenes are one of the largest classes of plant-derived natural products. These compounds protect plants against pests and diseases and they are also important as drugs and anticancer agents. Arabidopsis and cereals such as rice and maize do not make triterpene glycosides, which are associated with protection against pests and diseases. These plants were therefore excellent experimental systems for proof of concept experiments involving metabolic engineering of the triterpene pathway into heterologous species. The objective of our experiments was to advance in our understanding of triterpene biosynthesis, establishment of a customised toolkit of genes and enzymes for metabolic engineering of triterpene synthesis in plants, validation of this toolkit by heterologous expression and to advance in the methodology for multi-gene vector assembly. The expression vectors of different genes involved in triterpene biosynthesis were constructed using Golden Gate Assembly technique, which was chosen due to the complexity of the constructs and it allowed us to achieve an important advance in methodology for multi-gene vector assembly. Golden gate cloning toolkit was developed and it contains a set of domesticated regulatory and coding sequences being very useful for future experiments. We designed root specific as well as constitutive expression vectors and we introduced them in GV3101 Agrobacterium tumefaciens strain. We validated the toolkit in N. Benthamiana by single Agrobacteria culture, resulting in the production of the expected compounds from the avenacin metabolic pathway. Once the constructs were validated, we transformed Arabidopsis Col-0 by floral dip transformation method. T1 seeds were germinated in selection media containing kanamycin, timentin and nyastatin and resistant plants were transferred to soil and positive plants were selected by PCR. T2 plants were analysed by RT-qPCR for zygozity and copy number and T3 seeds from independent transformants were used for the analysis. An Arabidopsis line accumulating B-amyrin was regenerated, this line contained the 5' and 3' UTRs from the Cowpea mosaic virus and the mutated p19 gene silencing suppressor. Oat SAD1 and SAD2 gene under root specific and constitutive promoters were cloned and were transformed in rice and maize. Independent transformants were regenerated and analysed. Any of the regenerated lines accumulated the compound. Synthetic biology is a new important tendency in plant science and it will open new opportunities. We thought that it was a good opportunity to have a side project to make synthetic clusters containing combinations of genes that confer designer traits and introducing these into crops plants, where their expression can be co-ordinately controlled in responses to developmental, environmental or chemical cues (for instance, in the roots by nutrient limitation). This raises two important questions. Firstly, can functional clusters be built up from defined components? Secondly, are synthetic clusters able to be used for the introduction and controlling the expression of multi-gene designer traits (not necessarily restricted to metabolism) into crop plants? In our experiment, we made different synthetic clusters containing the coding sequence for the three dhurrin genes from Sorghum, each with the oat avenacin, the Arabidopsis thalional and 35S promoters and terminators (Table2). All the previous constructs were tested in N.Benthamiana prior Arabidopsis transformation. We obtained lines containing dhurrin under the expression of different promoters. The development of this tool kit has huge potential applications as we can develop a library containing different tissue-specific promoters including promoters that show responses to developmental, environmental or chemical cues such as pathogen, circadian clock, hypoxia stress, nitrate starvation and so on.

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