Stevens M Brumbley
University of North Texas, USA
Title: Metabolic engineering of a high biomass C4 grass for commercial scale production of bioplastics
Biography
Biography: Stevens M Brumbley
Abstract
Sugarcane is an important high biomass crop grown throughout the tropical and subtropical regions of the world. In Brazil, sugarcane has a central role in not only the production of sucrose but also in the production of biofuels and bio-based chemicals. In the future, these bio-based chemicals may be derived not only from bio-refineries but also from genetically engineered sugarcane itself. We have been metabolically engineering sugarcane to function as a bio-factory producing bio-plastics and bio-plastic precursors. Sugarcane was metabolically engineered to produce polyhydroxybutyrate (PHB), a member of the class of biopolymers known as polyhydroxyalkanoates (PHA). Most of our work has focused on PHB using a three enzyme pathway from a soil bacterium (Ralstoniaeutropha) [β-ketothiolase (PhaA), acetoacetyl-reductase (PhaB) and PHB synthase (PhaC)]. Transgenes were expressed in the nucleus of sugarcane and the enzymes were targeted to plastids. In plastids, using a maize polyubiquitin promoter, PHB granules were detected in all cell types in sugarcane stalks and leaves, however, the highest accumulation was in bundle sheath cells. A number of strategies were used to increase the levels of PHA accumulation in sugarcane leaf tissue. An early detection system was developed using Nile Blue. A staining and fluorescent microscopy showing that PHA could be detected in leaf tissue from tissue culture plantlets regenerated from the transgenic callus. When a stronger promoter was used (maize chlorophyll A/B binding protein promoter) PHA accumulation was significantly higher in leaf tissue of sugarcane. Knocking out competition for acetyl-CoA in sugarcane resulted in a 50% increase in PHB levels in leaves. Replacing PhaA with a novel enzyme resulted in another 4 fold increase in PHA levels resulting in leaves producing commercial levels of PHA. In addition to plastid production, PHB and PHB/PHA copolymers were engineered into sugarcane peroxisomes by targeting the PhaA, PhaB, and either PhaC or the Pseudomonas aeruginosa PhaC1 to peroxisomes. We found both PHB and PHB/PHA copolymer granules localized in peroxisomes and vacuoles in sugarcane leaves. The biopolymers we are producing have very high molecular weight with the highest at close to two million daltons. The highest production level of PHA polymer recorded to date in sugarcane is 12% of the total leaf dry weight.