Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

Effect of ionic liquid treatment on the structures of lignins in solutions: molecular subunits released from lignin.

The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low sulfonate (LS)--before and after treatment with 1-ethyl-3-methylimidazolium acetate were studied using small-angle neutron scattering (SANS) and dynamic light scattering (DLS) over a concentration range of 0.3-2.4 wt %. The results indicate that each of these lignins is comprised of aggregates of well-defined basal subunits, the shapes and sizes of which, in D(2)O and DMSO-d(6), are revealed using these techniques. LS lignin contains a substantial amount of nanometer-scale individual subunits. In aqueous solution these subunits have a well-defined elongated shape described well by ellipsoidal and cylindrical models. At low concentration the subunits are highly expanded in alkaline solution, and the effect is screened with increasing concentration. OS lignin dissolved in DMSO was found to consist of a narrow distribution of aggregates with average radius 200 ± 30 nm. K lignin in DMSO consists of aggregates with a very broad size distribution. After ionic liquid (IL) treatment, LS lignin subunits in alkaline solution maintained the elongated shape but were reduced in size. IL treatment of OS and K lignins led to the release of nanometer-scale subunits with well-defined size and shape.

Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass.

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.