Issued September 2008

Global Climate & Energy Project

STANFORD UNIVERSITY

Towards New Degradable Lignin Types

Investigators Wout Boerjan, Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent University Objective The long term goal of the project is to identify natural products (called ʻtarget moleculesʼ) that can be biosynthesized in energy crops, translocated through the plasma membrane and cross-coupled with lignin units such that the generated bonds 1) are more susceptible to chemical cleavage (Figure 1), or 2) prevent cross-linking with hemicellulose, or 3) render the lignin polymer more hydrophilic, or 4) can themselves be easily cleaved, or 5) a combination thereof. Ideally, the structures of the target molecules are very similar to traditional monolignols so that they can be exported to the wall using the same transport system. Background Lignin is an aromatic heteropolymer that negatively affects the quality of raw material for chemical pulping, forage digestibility and conversion to bioethanol. The polymer is built up by the combinatorial radical coupling of mainly coniferyl and sinapyl alcohol, although a range of minor units are also present in the polymer (Boerjan et al., 2003). Radical coupling results in a variety of chemical bonds, the frequency of which depends on the relative abundance of the various monomers, on the chemical characteristics of the monomers, and the local environment in the cell wall. For end use applications, such as conversion of lignocellulosic biomass to fermentable sugars in the process to bioethanol, cell walls would ideally contain less lignin. These lignin polymers should be rich in bonds that are easily cleaved enzymatically or chemically. Approach The project is divided into four tasks. Task 1 aims at defining which molecules are good targets for engineering and targeting to plant cell walls. This task will yield a ranked list of candidate molecules and is low risk.

Figure 1: Example of a target molecule.

BIO

MA

SS E

NER

GY

Issued September 2008

Task 2 aims at cloning biosynthetic genes for target molecules from exotic species and overexpressing these in bioenergy crops (Figure 2). Task 3 is more challenging, and aims at rerouting a selected number of target molecules, of which the biosynthetic pathway and subcellular localization are already partially known, to the cell wall in transgenic plants. Task 4 is the most risky, and aims at identifying biosynthetic pathways for promising target molecules by a combination of genetics and metabolomics. Some interesting target molecules have been detected only in some exotic species, and their biosynthetic pathways are entirely unknown. Engineering the biosynthesis of any of these molecules in energy crops, and routing them to the cell wall represents a daunting task. Task 4 aims at paving the way to simplify this task, starting from the hypothesis that some of the target molecules are not specific to the species in which they have been detected, but are also present in Arabidopsis. Only a fraction of the ~5000 molecules estimated to be present in Arabidopsis have a known identity. Hence, there is a significant possibility that the target molecules are present in Arabidopsis, but have not been identified in the right accession, tissue or condition. The strategy to be followed in this research activity is to search for these target molecules in seven tissues (stem, rosette, seed, petiole, root, flower, silique) of 300 Arabidopsis ecotypes using Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS) preceded by a linear ion trap. Genetical metabolomics and association genetics will then be used to identify biosynthetic genes involved in making these molecules.

Figure 2: Transgenic poplar trees as second generation energy crops. References Boerjan, W., Ralph, J., and Baucher, M. (2003). Lignin biosynthesis. Annu. Rev. Plant Biol. 54, 519-546.