Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Rima Menassa


Plants are wonderful living organisms. They are able to store solar energy into carbohydrates by fixing CO2 through photosynthesis which can be subsequently harvested and used for fuel production. However, one of the major limitations for transforming these carbohydrates into liquid fuels is the recalcitrance of the plant cell wall. Although microorganisms have evolved a series of cell wall degrading enzymes to harvest efficiently this energy and are considered the main source of these biocatalysts, harnessing these microorganisms for the production of enzymes is a costly process and a major factor limiting the commercialization of lignocellulosic biomass-to-ethanol processes. The production of cell wall degrading enzymes in plants has been proposed as an alternative platform to lower cost of production. However, due to the variability of biomass feedstock availability and plant cell wall complexity, specialized accessory enzymes are necessary for the complete plant cell wall deconstruction into fermentable sugars.

The expression of three key accessory enzymes was investigated in Nicotiana benthamiana. A new esterase gene from Acremonium alcalophilum was identified, produced in plants and characterized. It accumulated to high levels, 9% of total soluble protein (TSP), and it was found to have both acetyl xylan esterase and lipase activity. A polygalacturonase I from Aspergillus niger (AnPGI) and a feruloyl esterase from Anaeromyces mucronatus (Fae1A), were also produced using a combination of subcellular targeting and protein fusions with elastin-like polypeptide (ELP) and hydrophobin I (HFBI). Accumulation levels of 3.6, and 3.9% of TSP were achieved for AnPGI and Fae1A respectively. Based on our observations, although the use of protein fusions lowered protein activity, the ELP fusion had the most significant impact on enzyme production, with a higher amount of total activity recovered from fresh leaf weight than from HFBI fusions or unfused proteins. The impact of ELP and HFBI fusions on protein accumulation was protein-specific and should be evaluated individually in the future. The production of these three enzymes can lead to a minimal enzymatic cocktail for degrading the pectin component of the plant cell wall and should have an impact on the use of pectin-rich biomass residues for biofuel production.