Biofuels developed from biomass crops have the potential to supply a significant portion of our transportation fuel needs. To achieve this potential, however, it will be necessary to develop improved plant germplasm specifically tailored to serve as energy crops. Liquid transportation fuel can be created from the sugars locked inside plant cell walls. Unfortunately, these sugars are inherently resistant to hydrolytic release because they are contained in polysaccharides embedded in lignin. Overcoming this obstacle is a major objective toward developing sustainable bioenergy crop plants. The maize Corngrass1 ( Cg1 ) gene encodes a microRNA that promotes juvenile cell wall identities and morphology. To test the hypothesis that juvenile biomass has superior qualities as a potential biofuel feedstock, the Cg1 gene was transferred into several other plants, including the bioenergy crop Panicum virgatum (switchgrass). Such plants were found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment. In addition, a complete inhibition of flowering was observed in both greenhouse and field grown plants. These results point to the potential utility of this approach, both for the domestication of new biofuel crops, and for the limitation of transgene flow into native plant species.

Lignocellulosic biomass was used for thousands of years as animal feed and is now considered a great sugar source for biofuels production. It is composed mostly of secondary cell walls built with polysaccharide polymers that are embedded in lignin to reinforce the cell wall structure and maintain its integrity. Lignin is the primary material responsible for biomass recalcitrance to enzymatic hydrolysis. During plant development, deep reductions of lignin cause growth defects and often correlate with the loss of vessel integrity that adversely affects water and nutrient transport in plants. The work presented here describes a new approach to decrease lignin content while preventing vessel collapse and introduces a new strategy to boost transcription factor expression in native tissues. We used synthetic biology tools in Arabidopsis to rewire the secondary cell network by changing promoter-coding sequence associations. The result was a reduction in lignin and an increase in polysaccharide depositions in fibre cells. The promoter of a key lignin gene, C4H, was replaced by the vessel-specific promoter of transcription factor VND6. This rewired lignin biosynthesis specifically for vessel formation while disconnecting C4H expression from the fibre regulatory network. Secondly, the promoter of the IRX8 gene, secondary cell wall glycosyltransferase, was used to express a new copy of the fibre transcription factor NST1, and as the IRX8 promoter is induced by NST1, this also created an artificial positive feedback loop (APFL). The combination of strategies—lignin rewiring with APFL insertion—enhances polysaccharide deposition in stems without over-lignifying them, resulting in higher sugar yields after enzymatic hydrolysis.

SummaryLignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoylCoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C 6 C 1 lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C 6 C 1 monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.

Persil Çetinkol, Özgül (Dogus Author)In the biochemical conversion of lignocellulosic biomass to biofuels, the process of pretreatment is currently one of the most difficult and expensive operations. The use of ionic liquids (ILs) in biomass pretreatment has received considerable attention recently because of their effectiveness at decreasing biomass recalcitrance to subsequent enzymatic hydrolysis. In addition, ILs have the potential for decreasing the need for corrosive or toxic chemicals and associated waste streams that can be generated by other pretreatment methods that utilize acids and/or bases. In this article, we address two significant challenges to the realization of a practical IL pretreatment process. First, we describe a mixture containing specific proportions of a ketone and an alcohol that precipitates cellulose and lignocellulosic biomass from solutions of the IL 1-ethyl-3-methylimidazolium acetate without the formation of intermediate gel phases. Second, an IL recovery process is described that removes lignin and most residual IL solutes and that minimizes energy and solvent use. These two techniques are demonstrated by the pretreatment of 100 g of corn stover with the recovery of 89% of the initial IL and separate corn stover fractions rich in glucans, xylans, lignin, and non-polar substances. These results highlight one potential approach towards the realization of a scalable ionic liquid pretreatment process technology that enables ionic liquid recovery and reuse