Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues

Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the > 30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others.