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Oñate Carvajal, Felipe Alberto

University of British Columbia

Woody feedstocks, such as poplar and willow trees, are valuable renewable resources due to their rapid growth and non-competition with food crops. A key challenge in processing lignocellulosic biomass is the presence of lignin, a major component. Lignin typically consists of a polymer of three monolignols: p-coumaryl, coniferyl, and sinapyl alcohols. Efforts in genetic engineering poplar lignin have proven successful in altering the core lignin biosynthetic pathway, demonstrating its adaptability. Additionally, various phenolic compounds capable of integrating into lignin have been identified in different species. One such compound is p-hydroxybenzoate (pHB), found inherently in the lignins of poplar, willow, and aspen. This discovery led to the transgenic production of 3,4-dihydrobenzoate (DHB), which, unlike pHB, participates in radical coupling as well as acylating monolignols. Another compound that could potentially integrate into lignin is 3,4,5-trihydrobenzoic acid (THBA), also known as gallic acid, and is a potent antioxidant used in pharmaceuticals. My research explores the potential of producing 3,4,5-trihydrobenzoate from monolignol precursors in genetically engineered poplar trees, and analyzing the ensuing phenolic composition. In plants, THB production is hypothesized to be catalyzed by a shikimate dehydrogenase/dehydroquinate dehydratase (SDH/DHD) isoform. I engineered hybrid poplar (Populus alba x grandidentata) with the SDH/DHD gene (VvSDH3) from grape (Vitis vinifera) driven by a lignin-specific promoter. The resultant transgenic lines (VvSDH3-poplar) did not produce detectable THB in xylem methanolic extracts. To test if xylem-specific conditions hindered THB production, Arabidopsis thaliana plants were also transformed with the same VvSDH3 gene using a constitutive cauliflower mosaic virus 35S (CaMV35S) promoter, and again these plants did not yield detectable THB in leaf methanolic extracts. Given the absence of THBA production, an alternative biosynthetic pathway from Pseudomonas fluorescens was explored. A hydroxybenzoate hydroxylase encoded by the PobA gene, previously reported to hydroxylate pHBA and DHBA to THBA, was introduced into transgenic poplar trees producing pHBA or DHBA. These trees, grown in tissue culture and transformed with the PobA gene driven by either lignin-specific or cellulose-specific promoters, did not accumulate THBA. However, pHBA and DHBA were also not detectable, suggesting the tissue culture samples might not be suitable for assessing the production of THBA.

Text

2024-06-13

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88460

Forestry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/