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in tobacco leaves, transient expression of bacterial feedback-resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB) produces high titers of protocatechuate (PCA), which is in turn efficiently converted into 2-pyrone-4,6-dicarboxylic acid (PDC) upon co-expression of PCA 4,5-dioxygenase (PmdAB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (PmdC) derived from Comamonas testosteroni. Stable expression of AroG in Arabidopsis thaliana in a genetic background containing the QsuB gene enhances PCA content in plant biomass, presumably via an increase of the carbon flux through the shikimate pathway. Introducing AroG and the PDC biosynthetic genes (PmdA, PmdB, and PmdC) into the Arabidopsis QsuB background, or introducing the five genes (AroG, QsuB, PmdA, PmdB, and PmdC) stacked on a single construct into wild-type plants, results in PDC titers of about 1% and about 3% dry weight in plant biomass, respectively. Consistent with previous studies of plants expressing QsuB, all PDC producing lines show strong reduction in lignin content in stems. This low lignin trait is accompanied with improvements of biomass saccharification efficiency due to reduced cell wall recalcitrance to enzymatic degradation. Importantly, most transgenic lines show no reduction in biomass yields |
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