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UBC Theses and Dissertations

Engineering flavonoids into the lignins of poplar Mahon, Elizabeth Louise


Woody feedstocks, such as poplar, willow, and eucalyptus, represent abundant and fast-growing sources of lignocellulosic biomass for use in the production of pulp and paper, biofuels, and other bio-based materials. Lignin, a polyphenolic polymer, is the second most abundant chemical constituent of plant secondary cell walls and is typically composed of three canonical monolignols: p-coumaryl, coniferyl, and sinapyl alcohols. Recent efforts to genetically engineer the monolignol biosynthetic pathway have led to significant changes in the content and composition of lignin, highlighting the remarkable metabolic plasticity of this major biosynthetic pathway. Moreover, a wide array of non-traditional monolignols has been found to naturally incorporate into lignins of different plant species, such as the flavonoid tricin found in the lignins of grasses. To investigate the possibility of introducing flavonoids into the lignins of poplar as novel, value-added lignin monomers, I genetically engineered poplar to accumulate flavonoids in lignifying xylem tissue and analyzed the resulting wood chemistry. Chalcone synthase catalyzes the first committed reaction in the production of flavonoid compounds to produce naringenin chalcone which is then isomerized to naringenin. Using a lignin-specific promoter, I have genetically engineered hybrid poplar (Populus alba x grandidentata) to express a chalcone synthase gene (MdCHS3) derived from apple (Malus x domestica). MdCHS3-poplar accumulated naringenin in xylem methanolic extracts and NMR analysis revealed naringenin in the extract-free, cellulase-treated xylem tissue (enzyme lignin). MdCHS3-poplar displayed lower total lignin, an increase in cell wall carbohydrate content, and performed significantly better during saccharification assays compared to wild-type. Building on these promising results, I characterized two flavonoid-modifying enzymes derived from Brachypodium distachyon in vitro: chrysoeriol 5'-hydroxylase (BdCYP75B4) and flavone synthase II (BdCYP93G1), both key enzymes in the production of tricin and O-linked tricin glycosides. I also confirmed that PaxgOMT25, an important O-methyltransferase in monolignol biosynthesis, can participate in tricin biosynthesis. Co-expression of BdCYP75B4 and BdCYP93G1 in MdCHS3-poplar trees resulted in stunted growth and limited plant viability, however analysis of a recoverable low-expressing line revealed accumulation of tricin in xylem methanolic extracts not observable in controls, demonstrating that the successful production of tricin, a high value flavonoid, in poplar xylem is feasible.

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