Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

Fall 2020

Thesis Director

Thomas Canam

Thesis Committee Member

Eloy Martinez

Thesis Committee Member

Gopal R. Periyannan

Abstract

Lignocellulosic biomass is notoriously difficult to deconstruct into lignin, hemicellulose, and cellulose fractions without the use of biochemical, chemical, and/or mechanical pretreatments. These pretreatment strategies add operational cost and in some cases produce hazardous waste that poses health and safety risks. Direct biological pretreatment from biomass-degrading bacteria and fungi may significantly reduce the costs and risks associated with these traditional strategies due to the innate ability of these organisms to modify plant cell walls. An additional measure that could further reduce the expenses associated with lignocellulosic biomass fractionation is genetic modification of the lignocellulosic feedstocks, which can be accomplished by misregulating native genes and/or expressing foreign genes. In the present study, the genes for two hemicellulose-modifying enzymes, acetyl xylan esterase (AXE) and endo-polygalacturonase (EPG), were cloned from a white-rot fungus, Trametes versicolor, for the purposes of expressing these genes in plant systems. It was hypothesized that expression of these fungal enzymes in plants will reduce the covalent linkages in lignocellulose, particularly among the hemicellulose components of the cell wall. The AXE and EPG genes were subcloned into an entry plasmid, pDONR221, using Gateway technology, and were then transferred to binary plasmids (pEarleyGate backbones) suitable for Agrobacterium-mediated plant transformation. The genes are under the control of the 35S promoter with and without fluorescent protein fusions (CFP, YFP, or GFP). Future experiments will investigate the effects of these fungal hemicellulases on plant cell wall chemistry and digestibility in a variety of host systems (e.g. arabidopsis, tobacco, hybrid poplar), and explore the subcellular location of the enzymes using fluorescent microscopy.

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