Precision Genome and Metabolic Engineering for Genetic Improvement of the Biofuel Feedstock Sugarcane

Transcription activator-like effector nuclease (TALEN) is a recently developed tool enabling precise genome modifications, such as targeted mutagenesis, gene replacement, or insertion. Sugarcane is a prime feedstock for bioethanol production, and utilizing both sucrose and cell wall bound sugars for fermentation will enhance the biofuel yield. We recently demonstrated that RNAi mediated downregulation of lignin biosynthetic genes is a successful strategy to improve bioethanol production from lignocellulosic sugarcane biomass. In this study, one of the lignin biosynthetic genes, Caffeic acid O-methyltransferase (COMT) was targeted for the TALEN induced mutagenesis to modify lignin biosynthesis in sugarcane. Targeted mutations following TALEN delivery were identified by capillary electrophoresis of the COMT amplicon. Events were confirmed by sequencing of the COMT amplicon which revealed the presence of insertions and deletions at the target site. Data describing the total lignin content in the stem biomass of COMT mutants will be presented.

As an alternative to ethanol production, metabolic engineering might support accumulation of oil in vegetative sugarcane biomass for increased energy density and development of an advanced biofuel. Therefore, we are exploring the prospects of diverting the carbon flux from sucrose to triacylglycerol (TAG). This strategy involves the analysis of combinations of gene expression cassettes for metabolic engineering, supporting biosynthesis and storage of TAG. Correlations between TAG accumulation and gene combinations and their expression levels will be presented. These data indicate the feasibility of genetically engineering the high biomass crop sugarcane to produce TAG, which can be readily converted to biodiesel transportation fuel.