Engineering Microbial Surfaces to Produce Biofuels

Dwindling world supplies of petroleum have intensified the search for alternative sources of transportation energy. The production of ethanol and other commodities from sustainable biomass promises to reduce the world’s dependency on fossil fuels. A major obstacle to the commercialization of biofuels is the high cost of degrading biomass into fermentable sugars, which is typically achieved industrially through a two-step process in which the biomass is first thermochemically pretreated, then degraded by adding purified cellulase enzymes.  In principle, major reductions in costs as well as gains in efficiency may be achieved by using bacteria to degrade biomass instead of purified enzymes, or by creating a consolidated bioprocessor, a single microbe that has the capacity to convert lignocellulose into valuable end products such as ethanol.  Towards these objectives we have developed a robust protein display system that can be used to engineer the surface of Bacillus subtilis, a model organism that is highly amenable to genetic manipulation and well-suited for industrial application (29). Biofuels and many other high-value bio-based chemicals and materials can be produced from only twelve biomass-derived building blocks. We are currently working to introduce into B. subtilis the relevant metabolic pathways that produce these compounds, which, when paired with our established cellulose degrading system, could enable the direct production of many valuable biocommodities from biomass.

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Fig. 1. Cel8A is successfully displayed on the surface of B. subtilis. (A) Immunofluorescence micrographs of B. subtilis strain TDA03 dis- playing His6-tagged Cel8A. Left, cells of strain TDA02 expressing Cel8A. Middle, cells of strain TDA03 expressing only Cel8A. Right, cells of strain TDA03 expressing both SrtA and Cel8A. Cells were probed for the presence of Cel8A on the surface with mouse anti-His6 serum and fluorescently stained anti-mouse IgG conjugated to Dylight- 488. 4,6-diamidino-2-phenylindole (DAPI) was used to stain the DNA. In images containing larger numbers of cells, a similar display pattern was observed.