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Bio-energy

Bio-Energy and Bio-Aromatics

Wout Boerjan

woboeIt is now well recognized that burning fossil fuels and deforestation are major contributors to climate change, and that 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. As lignin needs to be depolymerized for both purposes, we focus on understanding the biosynthesis, polymerization and structure of lignin, and how lignin biosynthesis integrates into plant metabolism and development. 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 is difficult to purify them for structural elucidation by NMR. The group has a major activity in characterizing these metabolites. Arabidopsis, maize and poplar are used as a model species for gene and metabolite discovery. Field trials are made to investigate the new traits in a relevant environment..

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. From co-expression data sets, we have identified a set of candidate genes that likely play an important role in phenolic biosynthesis. We have already demonstrated the role of some of these in lignin biosynthesis (Vanholme et al., 2012; 2013; Sundin et al., 2014). 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, and by translating this research to maize and poplar. Double mutants are made to test for additive or synergistic effects (de Vries et al., 2018).
 
Arabidopsis co-expression network.
 
Arabidopsis stem pieces of a wild type plant (left) and a low lignin mutant (right) after treatment with cellulases.

Engineering plants for reduced lignin amount or altered structure is often associated with a yield penalty. Several hypotheses exist that explain the molecular basis of the yield penalty. First, it has been shown that lignin modification results in a collapse of vessels (irx phenotype) (Yang et al., 2013). We have shown that the lignin reduction in ccr and cse mutants results in dwarfism, and that restoring lignin in the xylem vessels restores growth (Vargas et al., 2016; De Meester et al., 2018). We are examining whether a similar strategy can be used to overcome the yield penalty in poplar.
 

The figure at the left shows that the biomass penalty of low-lignin mutants can be overcome by restoring lignin biosynthesis in the vasculature. Wild-type Arabidopsis (left), the low lignin ccr1 mutant (middle), and the vessel-complemented ccr1 mutant (right). 

A second plausible cause of the yield penalty is that soluble phenolics with bio-activity may differentially accumulate in the lignin-modified plants compared to the wild type. For example, trans-cinnamic acid, the substrate of C4H at the entry point of phenylpropanoid biosynthesis, is isomerized to cis-cinnamic acid, which we have shown to act as an auxin-transport inhibitor (Steenackers et al., 2017).

The monolignol biosynthesis precursor trans-cinnamic acid is converted to cis-cinnamic acid by UV light. Addition of cis-cinnamic acid to the growth medium promotes lateral root formation in Arabidopsis.

In addition to engineering lignin structure by genes of the host plant itself (modification of H/G/S/benzodioxanes/aldehydes/ferulates), it might as well be 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) and exemplified by Wilkerson et al. (2013). In this approach, the host plant is transformed with (a) heterologous gene(s) that encode(s) (a) biosynthetic enzyme(s) that is(are) able to make a monolignol substitute. When transported to the cell wall and incorporated into the lignin polymer, this molecule generates a bond that is more susceptible to the biomass pretreatment that is used to degrade the lignin polymer.

A main bottleneck in our gene discovery studies is that the identity of most metabolites is unknown. We can only discover the function of new genes encoding metabolic steps if 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 maize stems and leaves, the focal tissues in our reverse genetic analyses. To this end, we use the CSPP algorithm that was developed in the group and that is used 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 is done 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.

Our expertise in metabolite profiling of secondary metabolites has allowed to establish a VIB Metabolomics Core facility.

CSPP network of metabolites from maize.

 

A major objective of the group is to demonstrate, in field trials, that it is possible to engineer biomass crops such that the biomass processing is improved without associated yield penalty. 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. 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.

Field trial of popular genetically engineered to produce lower amounts of the CINNAMYL ALCOHOL DEHYDROGENASE (CAD1) protein, an enzyme involved in lignin biosynthesis.

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 said to be 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.

Lignin research contributes to the circular bio-based economy, and thereby combats climate change. More details here.

Another example is the conversion of plant biomass into a range of products that are nowadays mainly 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 plant biomass-derived products has an ecological footprint and is limited by the growth speed of the plants. Therefore, also products derived from plant biomass should not be spilled or over-consumed.

Taken together, research in the "Bio-energy and Bio-aromatics" group aims at supporting the transition from a fossil-based to a bio-based economy.

Team building 2018.

Lees het nut van lignine-onderzoek in het Nederlands hier.

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