Development of the thermally tolerant pectin methylesterase for an innovative application in biomass processing

Sugar beets (Beta vulgaris L.) are the major source of food-grade sucrose produced in the United States. Beet sugar production is energy intensive and generates high greenhouse gas emissions. Up to a third of the energy consumed in a processing factory is used to dry beet pulp, the pectin-rich biomass left after sucrose extraction. Previous studies investigated the thermally tolerant pectin methylesterase (TT-PME; EC 3.1.1.11) for its role in degrading citrus juice quality and for modifying pectin for food applications. My research investigated TT-PME for an innovative application in processing pectin-rich biomass. This addressed the overall hypothesis that selective action by thermally tolerant pectinases such as TT-PME, can modify cell wall structure to reduce energy demands for drying beet pulp. The Specific Aims were to: 1) evaluate pectinase treatment of sugar beet pulp for improved processing properties, 2) generate selective antibodies for differentially identifying recombinant TT-PME expressed in plant tissues, and 3) determine functionality of TT-PME's pre and PRO regions for recombinant expression in planta by transient expression. To test the hypothesis, I developed a new assay to quantify water binding by beet pulp following enzyme treatments. Thus, I determined PME treatment reduced water binding in beet pulp by 25% through a calcium-mediated mechanism. Additional treatments with arabinanase (E.C. 3.2.1.99) showed no effect on water separation. These results provided proof of concept that TT-PME expression in sugar beets may confer a processing benefit by facilitating water separation from beet pulp. For TT-PME expression studies in transgenic plants, antibodies for TT-PME were produced from a sequence-specific peptide. These antibodies differentially detected TT-PME from other plant PMEs. Towards developing an expression strategy for sugar beets, TT-PME structural constructs were tested in the Nicotiana benthamiana transient expression system. TT-PME's native signal peptide yielded high expression levels, equal to the well-established patatin signal peptide. Furthermore, I demonstrated that TT-PME must be expressed with its PRO region. These results will direct future TT-PME expression studies in biotech beets to deliver the benefit of reduced processing energy demands. These TT-PME expressing biotech beets will improve economic profitability while reducing the carbon footprint of beet sugar factories.