15-02: Engineering switchgrass CAD

Lignin is complex heteropolymer and an important component of plant cell walls that helps provide structural integrity and disease resistance.  However, it also increases biomass recalcitrance to hydrolytic enzymes, which reduces overall process yields.  Lignin is synthesized by a suite of at least twelve enzymes, although knowledge is incomplete on how metabolic fluxes through the pathways are controlled as well as mechanisms governing enzyme substrate preferences and kinetics.  Previously, we characterized two genes from switchgrass (PviCAD1 and PviCAD2) that encode for a key enzyme involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase (CAD).  Our investigations focused on two objectives: (1) finding key residues that impact CAD activity and substrate preferences and (2) understanding the impact of CAD down-regulation in switchgrass.  To satisfy objective 1, several CAD mutants were generated using site-directed mutagenesis of residues that were of known or suspected importance to catalytic activity or substrate docking.  Purified recombinant proteins displayed distinct differences in their kinetic properties compared to controls and results showed that changing one or two residues was sufficient to dramatically alter CAD activity.  In order to investigate objective 2, RNAi techniques were used to silence CAD through Agrobacterium mediated transformation.  The transformants contained lower levels of CAD RNA transcript and CAD protein.  Furthermore, plant protein extracts displayed lower levels of CAD activity against the monolignol substrates coniferaldehyde and sinapaldehyde.  CAD down-regulation also lowered overall plant lignin content and increased glucose release in pretreated plant samples.