Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

Ayalew Ligaba-Osena, Jenna Jones, Emmanuel Donkor, Sanjeev Chandrayan, Farris Pole, Chang-Hao Wu, Claire Vieille, Michael W. W. Adams, Bertrand B. Hankoua

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase fromPyrococcus furiosusand glucoamylase fromSulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) fromEscherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due toglgCexpression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activityin vitrothan the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes producedin plantamaintain their activity and thermostability.