Abstract
Acetic acid bacteria have unique metabolic characteristics that suit them for a variety of biotechnological applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and enantioselective partial oxidations 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. Expression of extracytoplasmic enzymes to augment the oxidative capabilities of acetic acid bacteria is desired but is challenging due to the already crowded inner membrane. 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. Outer membrane porin F (OprF) was used to deliver alkaline phosphatase (PhoA) to the cell surface. Constitutive high-strength p264 and moderate-strength p452 promoters were used to direct expression of the surface display system. This system was demonstrated for biocatalysis in whole-cell assays with the p264 promoter having a twofold increase in PhoA activity compared to the p452 promoter. Proteolytic cleavage of PhoA from the cell surface confirmed proper delivery to the outer membrane. Furthermore, a linker library was constructed to optimize surface display. A rigid (EAAAK)1 linker led to the greatest improvement, increasing PhoA activity by 69%. This surface display system could be used both to extend the capabilities of acetic acid bacteria in current biotechnological processes, and to broaden the potential of these microbes in the production of value-added products.
Highlights
Gluconobacter oxydans is an industrially important microbe belonging to the family Acetobacteriaceae, commonly referred to as the acetic acid bacteria
Surface display in G. oxydans To enable surface display in acetic acid bacteria, a truncated version of Outer membrane porin F (OprF) (OprF188) was tested for its ability to localize the PhoA reporter enzyme to the cell surface of G. oxydans
The OprF188-PhoA surface display systems produced statistically significant absorbance changes compared to strains expressing the anchor protein alone when using both the high- (q < 0.001) and moderate-strength (q < 0.001) promoters, p264 and p452, respectively
Summary
Gluconobacter oxydans is an industrially important microbe belonging to the family Acetobacteriaceae, commonly referred to as the acetic acid bacteria. G. oxydans is an incomplete oxidation specialist known for its ability to partially oxidize alcohols, polyols, and monosaccharides to produce a diverse array of aldehydes, ketones, and organic acids. These reactions occur in the periplasmic space, as G. oxydans possesses an arsenal of dehydrogenases bound to the inner membrane. The membrane-bound dehydrogenases are both regiospecific and enantioselective, allowing the microorganism to produce chiral compounds from precursors containing multiple identical functional groups, such as sugars and polyols. G. oxydans is osmotolerant and acidophilic, which are desirable industrial characteristics (Olijve & Kok, 1979)
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