Bio-Energy and Bio-Aromatics

The use of fossil fuels is a major contributor to climate change. In contrast, plant biomass can serve as a renewable and potentially carbon-neutral raw material for the production of bioenergy and a variety of other biobased products. The major long-term goal of the Bioenergy and Bio-aromatics group is to engineer plant cell wall composition for a more cost-effective conversion of plant biomass into fermentable sugars or aromatic building blocks, without adversely affecting plant yield. Because the cell wall polymer lignin plays a central role in these conversion processes, we focus on understanding the biosynthesis, polymerization and structure of lignin, and use this information to tailor plants with new properties. Field trials are established to investigate the new traits in a relevant environment. We also study how lignin biosynthesis integrates into plant metabolism and development by investigating the potential bioactive properties of metabolites that are part of -or derived from- the lignin biosynthesis pathway. In addition to lignin and cell wall polysaccharides, plant biomass also contains thousands of molecules of which the structures, and hence the properties, have remained unknown for the simple reason that it is difficult to purify them for structural elucidation by NMR. We have a major activity in characterizing these metabolites alongside with their biosynthetic pathways and genes by integrating mass spectrometry, systems biology, GWAS and reverse genetics. We use Arabidopsis, maize and poplar as model species.
Identifying new genes in aromatic metabolism
When improving plant cell walls, it all comes down to identifying the genes that are involved in the biosynthesis of the major cell wall polymers, and altering their expression levels in target crops such as poplar and maize. Through co-expression analyses in Arabidopsis and maize, we have identified a set of candidate genes that likely play an important role in phenolic biosynthesis. We have already demonstrated the role for some of these in the biosynthesis of lignin and coumarins (Vanholme et al., 2013; Sundin et al., 2014; Vanholme et al., 2019). Our expertise in comparative metabolite profiling and mass spectrometry is a great asset to help elucidating their function. The potential for applications of these genes is investigated by analysing the biomass composition and saccharification potential of the corresponding mutants in Arabidopsis, poplar and maize. Double mutants are made to test for additive or synergistic effects (e.g. de Vries et al., 2018).
Overcoming the yield penalty of lignin-modified plants
hypotheses have been put forward to explain the molecular basis of the yield penalty. It has been shown that lignin modification often results in a collapse of vessels, which negatively affects water and nutrient transport in the plant. We have shown that the lignin reduction in ccr and cse mutants results in smaller plants, and that restoring lignin in the xylem vessels restores growth (Vargas et al., 2016; De Meester et al., 2018). Another strategy to avoid growth phenotypes is to engineer weak alleles that subtly reduce enzyme activity, as we have demonstrated by engineering the combination of a null and a haploinsufficient CCR2 allele in poplar (De Meester et al., 2020).
Altering the structure of lignin by engineering monolignol substitutes
In addition to engineering lignin structure by using genes of the host plant itself (modification of H/G/S/benzodioxanes/aldehydes/ferulates levels; Chanoca et al., 2019), it is also possible to engineer easily degradable lignin polymers by using genes from other taxa in a synthetic biology approach, as discussed in Vanholme et al. (2012). In this approach, the host plant is transformed with one or multiple heterologous genes that encode biosynthetic enzymes that are able to make a monolignol substitute. When translocated to the cell wall and incorporated into the lignin polymer, this alternative monomer generates a bond that is more susceptible to the biomass pretreatment that is used to degrade the lignin polymer. By heterologous expression of two genes from Curcuma longa, we have been able to engineer Arabidopsis plants that incorporate diferuloyl methane (curcumin) into their lignin, thereby making the new lignin more susceptible to alkaline pretreatments (Oyarce et al., 2019).
Bioactive phenylpropanoids
A second plausible cause of the observed phenotypic consequences of lignin-modified plants is the differential accumulation of soluble bioactive phenolics in the lignin-modified plants compared to the wild type. To investigate the role of bioactive phenylpropanoids in plants, we primarily focus on cis-cinnamic acid and coumarin, using Arabidopsis as model system. cis-Cinnamic acid is the UV-isomerization product of trans-cinnamic acid, which is positioned at the entry point of the phenylpropanoid pathway. Our research demonstrated that cis-cinnamic acid acts as an auxin transport inhibitor and can be used as a natural plant growth-promoting compound (Steenackers et al., 2017; 2019).
Systematic identification of secondary metabolite structures in Arabidopsis and biomass crops
A main bottleneck in our gene discovery studies is that the identity of most metabolites is unknown. We can only understand the response to pathway perturbations, when we know the identity of the differentially accumulating compounds, e.g. in reverse genetics studies where we aim at identifying the substrate for an enzyme. One major objective in the group is to systematically identify the main secondary metabolites in Arabidopsis, maize and poplar, the focal species in our reverse genetic analyses. To this end, we use the LCMS-based CSPP algorithm that we developed in our team and that we use to characterize unknown compounds (Morreel et al., 2014). The algorithm searches for peak pairs that differ by a mass that corresponds to an enzymatic reaction. If this search is performed for all peaks in a chromatogram, and for the most prominent reactions that take place in metabolism, self-propagating networks are generated where each node is a metabolite and each edge a metabolic conversion. At the same time, the algorithm also predicts tentative biosynthetic pathways. Together with Mass Spectral Metadata analysis, an approach that combines data from different MS instruments and/or MS methods, CSPPs allowed the construction of a secondary metabolome database for maize (DynLib database (https://bioit3.irc.ugent.be/dynlib/); Desmet et al., 2021). In the ERC project ‘POPMET - Large-scale identification of secondary metabolites, their biosynthetic pathways and their genes in the model tree poplar’(https://cordis.europa.eu/project/id/834923), we integrate mass spectrometry, systems biology, GWAS and reverse genetics to identify new genes in secondary metabolic pathways in poplar.
 
Our expertise in metabolite profiling of secondary metabolites has allowed establishing the VIB Metabolomics Core facility (https://corefacilities.vib.be/metabcore).
Translational research in bio-energy crops
A major objective of the group is to demonstrate, in field trials, that the improved biomass processing efficiency of the engineered, greenhouse-grown biomass crops is maintained when plants are grown in the field. This is especially relevant for poplar, as trees are harvested in winter when leaves have been shed and the wood formation program has ended. A second objective of field trials is to assess the potential negative effects of lignin engineering on yield. Third, field trials allow harvesting enough biomass to test processing efficiencies at semi-industrial scale. Our previous field trial with CCR downregulated poplar showed that we can significantly improve biomass processing, but not yet to the level that it is useful for the biorefinery (Van Acker et al., 2014). In addition, the downregulation of CCR was variable and associated with a yield penalty. The objective now is to stably engineer plants with modified lignin, but that do not have a yield penalty, by making use of the CRISPR/Cas9 technology, and to evaluate these plants (poplar/maize) in experimental field trials.
One limitation with the use of CRISPR/Cas9 in poplar is that in order to establish field trials, the CAS9 gene needs to be eliminated from the genome-edited trees. Given that segregation is no option in trees and a second transformation is cumbersome, we are in the process of establishing a DNA-free gene-editing protocol for poplar.
Relevance of lignin research for a more sustainable society
Plants use sunlight and water to capture CO2 from the atmosphere to build their biomass. That biomass can then be converted to products that are nowadays made from fossil resources such as petroleum. Whereas the use of fossil resources leads to a net increase of CO2 into the atmosphere (e.g. when burning it as fuel), this is not the case when using plant biomass. For this reason, plant biomass is considered a renewable, carbon-neutral resource.
Lignin research is highly relevant for a number of applications. One example is the production of paper. In order to make paper from wood, lignin needs to be extracted from it. This extraction process involves cooking of the wood chips in strong alkaline conditions at high temperatures. Wood derived from trees that produce less lignin can be converted to paper using less chemicals and energy, which is positive for the environment.
Another example is the conversion of plant biomass into a range of products that are nowadays made from petroleum, such as fuels, plastics, detergents, etc... In this process, the cellulose is broken down into glucose units by enzymes. The glucose units are then fed to micro-organisms that ferment the glucose into products that are useful for society. Also for this application, lignin needs to be extracted from the biomass, because it prevents access of the enzymes to the cellulose. When the lignin levels are low, the conversion of plant biomass is more efficient.
A third field of interest is the improvement of the digestibility of fodder crops by ruminants. Fodder crops that produce less lignin are more easily digested by ruminants, allowing the production of more meat and milk per acreage. The production of meat has a high impact on the environment. It is therefore even much better to reduce our meat consumption such that more land can be set aside for other purposes such as afforestation and reforestation.
A fourth field of valorisation of lignin research is the production of aromatic molecules from lignin itself. Lignin can be depolymerized by catalytic reduction or pyrolysis into simple phenolic molecules that can be used as building blocks for the chemical industry. Also in this case, the use of plant biomass is a renewable alternative for the use of fossil resources.
Importantly, the plants used as feedstock for the biorefinery should be grown in a sustainable manner, in which forests and fields support biodiversity and the well-being of residents and visitors. Even though plant biomass is essentially renewable, the production of biomass-derived products has an ecological footprint and is limited by the growth speed of the plants. Therefore, also products derived from biomass should not be spilled or over-consumed.

Read more Lees meer

Publications

De Meester, B., Madariaga Calderón, B., de Vries, L., Pollier, J., Goeminne, G., Van Doorsselaere, J., Chen, M., Ralph, J., Vanholme, R., Boerjan, W.

Resources