Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.
(A) Schematic representation of the biosynthetic pathways for anthocyanins and proanthocyanidins. Abbreviations: chalcone synthase (CHS); chalcone isomerase (CHI); dihydroflavonol reductase (DFR); flavanone 3-hydroxylase (F3H); flavonol synthase (FLS); leucoanthocyanidin reductase (LAR); anthocyanidin synthase (ANS); anthocyanidin reductase (ANR); uridine diphosphate glucose-flavonoid 3-O-glucosyl transferase (UFGT). Purple colored arrows represent the catalytic steps that are upregulated by the overexpression of the A. majus transcription factors Rosea1 and Delila. The yellow highlighted area indicates the catalytic steps that are overexpressed in this work by the M. truncatula genes introduced in our multigenic construct, whose catalytic steps are highlighted in darker yellow and blue. (B) Premade GBParts, Modules and vectors used in this work. This includes the parts pCaMV35S promoter (GB0030), pTNos (GB0035), three vectors of the pGreenII-based pDGB1 series (Alfa1, Alfa2 and Omega2), one vector of the pCAMBIA pDGB2 series (Omega1) and two preassembled modules that were previously tested by the GB2.0 developers. The first module (GB0129) expresses the two A. majus transcriptional factors Rosea1 and Delila that under the control of the CaMV35S promoter. The second module (GB0235) is the hygromycin resistant cassette that is used to select the transformed plants in the stable transformation process. CaMV35S is the Cauliflower Mosaic Virus 35S Promoter; TNos is the Nopaline synthase terminator; PNos is the Nopaline synthase promoter; KR and SR stand for bacterial kanamycin and spectinomycin resistance cassettes; LB and RB represent the Left and Right Borders of the T-DNA. (C) GoldenBraid 2.0 multigenic construct AmRosea1:AmDelila:MtANR:MtLAR generated in this work. The multigenic construct was generated in five steps that include the assembly of the MtANR and MtLAR transcriptional units from its basic parts (Assemblies 1 and 2), the combination of these transcriptional units in a single vector (Assembly 3), the later addition of the A. majus transcriptional factors to the M. truncatula genes (Assembly 4) and finally the incorporation of the hygromycin resistance cassette to generate the multigenic construct that is used in all the experiments of this work. MtANR is the M. truncatula anthocyanidin reductase gene; MtLAR is the M. truncatula leucoanthocyanidin reductase gene.