Metabolic engineering of C₄ grasses for biofuel applications

Abstract

The amount of (1,3;1,4)-β-D-glucans or mixed-linkage-β-glucans (MLG) in biofuel crops is of interest because this is a source of fermentable hexose sugar. Recent modification of (1,3;1,4)-β-glucan in barley has opened the possibility of increasing the (1,3;1,4)-β-glucan amount in other plants, including bioethanol crops such as sorghum, a C₄ grass. As S. viridis is a model plant for studying C₄ biofuel crops the characteristics of MLG accumulation in S. viridis is of interest. The initial study determines the levels, structure and distribution of MLG in various vegetative tissues and grain in S. viridis, and investigates the relationship with the transcript levels of Cellulose synthase-like (Csl) genes, i.e. genes involve in MLG synthesis. Modification of MLG amount was tested by generating transformants of S. viridis with over-expressing Csl F6 driven by either the oat globulin promoter (ProASGLO) or the constitutive 35S promoter (Pro35S) from barley (HvCslF6) and sorghum (SbCslF6). In addition, a previous study on the over-expression of the (1,3;1,4)-β-glucan synthase in transgenic barley showed an excessive deposition of the polysaccharide in many cell types causing vascular suffocation and sometimes plant death. To resolve this problem this study also aim to identify a sorghum promoter which may be used to regulate the over-expression of (1,3;1,4)-β-D-glucan synthase genes (such as CslF and CslH) and restrict it to certain specific cell types only. Five putative sorghum mesophyll cell-specific promoters from genes involved in C₄ photosynthesis have been fused with a β-glucuronidase (uidA) cDNA. All modifications were tested in planta using stable Agrobacterium-mediated transformation.