AN EXAMINATION OF THE STRATEGY TO FACILITATE THE CONVERSION OF LIGNOCELLULOSE TO BIOETHANOL VIA LIGNIN MODIFICATION
Abstract:
This thesis focuses on developing a novel strategy to increase the digestibility of lignocellulosic biomass in bioethanol production by introducing peptides to the plant cell wall. First, the present study tested an experimental approach to verify the subcellular localization of apoplastic signal peptides identified by blasting and proteomic study in poplar. Second, the present study characterized the chemistry of cross linking between amino acids and proteins with lignin. Third, poplar was transformed to express and secrete proteins into the lignifying cell wall. The digestibility of most transgenic lines was increased compared to wildtype. In total, eight signal peptides were tested for protein subcellular localization using enhanced green fluorescence protein (EGFP) as a reporter. One of the signal peptides is from a putative Populus coniferin-β-glucosidase (CβG), which was first identified by blasting a cell wall-localized protein, lodgepole pine (Pinus contorta) CβG, against the Populus trichocarpa genome. The other seven signal peptides were selected from a poplar xylem sap proteome study. Eight signal peptides were inserted into EGFP-containing vector for plant transformation. Transformants of onion epidermal cells from the eight signal peptides showed extracellular localization of EGFP, however at different ratios comparing to total EGFP fluorescence intensity. Populus CβG was first identified in this study and fasciclin-like arabinogalactan protein 10 precursor (AGP) exhibited the highest extracellular to total EGFP intensity ratio in onion. Accordingly these two were also transiently transformed to native plant, hybrid poplar (Populus deltoides×nigra). In the transformants, EGFP was detected in the endoplasmic reticulum (ER) secretion pathway and extracellular space. Our experimental results are highly consistent with proteomics database and this study provides a fast and quantitative way for detecting extracellular localization of apoplastic proteins. Our results show that three amino acids (Cys, Tyr, and Thr) are able to react with intermediates of in vitro lignin synthesis: incubations of coniferyl alcohol, peroxidase and H2O2. LC/MS results show that the mechanism of cross linking of Cys, Tyr and Thr with lignin, is through the quinone methide intermediate of synthesis. In addition to free amino acids, our results also show the ability of whole proteins to cross link with lignin in vitro through Cys or through Tyr residues. Our findings provide a mechanism by which proteins and lignin can cross link in the plant cell wall and reveal the chemical nature of the interactions between protein and lignin. Poplar was transformed to express and secrete proteins into the lignifying cell wall. One construct (NTRG2) contains a gene encoding a peptide with high Tyr content (17%) with the poplar phenylalanine ammonia lyase (PAL2) promoter (Gray-Mitsumune et al. 1999) and a signal peptide from Pinus coniferin β glucosidase (CβG) (Samuels et al. 2002). The other vector (MGRP) has a gene encoding a peptide rich in Cys (11% Cys), which is able to participate in cross linking reactions with lignin in vitro. This vector is driven by the double 35S CaMV promoter and poplar CβG signal peptide. Transgenic poplars of NTRG2 showed normal plant growth, morphology, stem structure, and unaltered cell wall composition. Transgenic poplars of MGRP showed slow plant growth, narrow leaf shape, and small stem radius. The digestibility of most transgenic lines of NTRG2 and MGRP surveyed was increased compared to wildtype.
AN EXAMINATION OF THE STRATEGY TO FACILITATE THE CONVERSION OF LIGNOCELLULOSE TO BIOETHANOL VIA LIGNIN MODIFICATION. GET MORE PLANT BIOLOGY PROJECT TOPICS AND MATERIALS IN NIGERIA