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. 2016 Oct 26;7:13215. doi: 10.1038/ncomms13215

Figure 2. Assembling multi-gene pathways into pYB vectors with jStack.

Figure 2

(a) Stacking various reporter genes together into one transfer DNA: β-glucuronidase (GUS), GFP, DsRed. Promoters driving each gene are labelled and denoted by black arrows in front of coding sequences. Stable soybean root transformant lines were generated via Agrobacterium rhizogenes. Transformed roots exhibited GUS activity and GFP and DsRed fluorescence. Scale bar represents 20 μm. (b) Optimizing engineered metabolic pathways of a potential biofuel with gene stacking. Stacking various upstream enzymes in conjunction with new organellar targeting increased Bisabolene yields in planta—values are labelled in the graph. By stacking upstream enzymes such as chloroplast-localized farnesyl pyrophosphate synthase (CpFPPS) and chloroplast-localized 1-Deoxy-D-xylulose 5-phosphate synthase (CpDXS) with either cytosolic or plastid-targeted bisabolene synthase (BisSyn and CpBisSyn, respectively), increased yields of bisabolene were observed. Error bars indicate standard deviation, n=3. Mevalonate (MVA), isopentenyl pyrophosphate (IPP), farnesyl pyrophosphate (FPP), pyruvate (Pyr), glyceraldehyde 3-phosphate (G3P). (c) Engineering heterologous bacterial metabolites into plants. The cytotoxic molecule violacein is found in various soil bacteria and requires five enzymes (VioA, VioB, VioC, VioD and VioE) for its biosynthesis from tryptophan. All the five genes were stacked via yeast assembly and infiltrated into Nicotiana benthamiana leaves. Violacein production was observed via LC-MS methods.