Date of Graduation

Spring 2017

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Paul Schweiger

Keywords

Gluconobacter oxydans, surface display, biocatalysis, fusion linkers, outer membrane proteins

Subject Categories

Biology

Abstract

Acetic acid bacteria (AAB) have unique metabolic characteristics that suit them for a variety of applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and enantioselective partial oxidation of sugars, alcohols, and polyols. The resulting products are deposited directly into the medium where they are easily recovered for use as pharmaceutical precursors, industrial chemicals, food additives, and consumer products. Unfortunately, there has been little research towards improving AAB, as few molecular tools exist for metabolic engineering of these microbes. To this end, an original surface display system was developed to express recombinant enzymes at the outer membrane of the model acetic acid bacterium Gluconobacter oxydans. Three anchor proteins (OprF188, INPNC, and PgsA) were tested for the ability to deliver alkaline phosphatase enzyme, PhoA, to the cell surface. The OprF188 system was demonstrated for biocatalysis in whole-cell assays, and PhoA was proteolytically cleaved from the cell surface, suggesting proper delivery to the outer membrane. A linker library was also constructed to optimize surface display. The (EAAAK)1 rigid linker led to the greatest improvement, increasing PhoA activity by 70%. Surface display could be used both to extend the capabilities of AAB in current biotechnological processes, and to broaden the potential of these microbes in the production of value-added products.

Copyright

© Marshal Allen Blank

Open Access

Included in

Biology Commons

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