The native cellulolytic organisms, e. g., C. thermocellum, are a starting point for developing anaerobic microbes capable of one-step processing of cellulosic biomass to ethanol or other desired products in the absence of added saccharolytic enzymes (12, 15, 28). However, it is necessary to perform metabolic engineering to improve product yield and titer relative to performance obtained to date with traditional ethanologenic strains. Substantial advances have been made in developing the genetic transformation tools for cellulolytic bacteria (58, 62, 70, 71). Recently, metabolic engineering has been applied to a mesophilic, cellulolytic C. cellulolyticum to achieve high ethanol yields. This was achieved by heterologous expres­sion of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase (70). The fermentation pattern was shifted significantly in that ethanol production increased by 53%, acetate increased by 93%, and lactate decreased by 48%.

From a fundamental viewpoint, it is also interesting to study the mechanisms of hydrolysis product uptake by natural cellulolytic microorganisms, determine hydrolysis product con­centrations on the cell surfaces, and study the relationships between sugar transportation, energy level, and cellulase regulation.

From a practical viewpoint, another concern about naturally cellulolytic microorganisms is their sensitivity to the final product accumulation. For example, the wild C. thermo­cellum strain cannot grow in the presence of ethanol concentrations above 1% (w/v), yet industrial yeasts can tolerate ethanol titers up to 10% (w/v). Currently, an ethanol-adapted C. thermocellum strain that can tolerate up to 5.0-8.0% (w/v) ethanol was selected by step­wise increase in ethanol concentration (72). Another new method is global transcription machinery engineering (gTME), which re-programs gene transcription to tolerate substrate and product inhibition (73). Using gTME, a recombinant S. cerevisiae increased volumetric ethanol production by 69%, specific productivity by 41%, and ethanol yield by 14% (73). We expect that this technology could be applied to natural cellulolytic microorganisms as well.

The key objective for effective, recombinant cellulolytic ethanologens, e. g., S. cerevisiae, is the introduction of an efficient cellulase enzyme system. Key objectives to enable CBP microorganisms that we learned from natural cellulolytic microorganisms are to: 1) in­crease active recombinant cellulase expression levels up to 2-20% of cellular proteins (28), 2) optimize the expressed cellulase ratio (endo-/exo-/BG) by gene regulation, depending on their turnover number and substrate characterization (74), 3) generate more ATP from intracellular substrate phosphorylation by introduction of recombinant cellobiose and cel- lulodextrin phosphorylases, especially for anaerobic fermentations (44, 49), and 4) form an enzyme-microbe complex to reduce the enzyme synthesis burden by taking advantage of enzyme-microbe synergy (55).

Acknowledgment

Y. H.P. Zhang wishes to thank the Biological Systems Engineering Department of Virginia Polytechnic Institute and State University. Funding for this work also came from the USDA — CSREES 2006-38909-03484 grant and the DOE BSE and NIST.

[1] A genetic screen led by Reiter et al. (125) identified the mur4 mutant in Arabidopsis that has a 50% reduction of arabinose in the wall. The encoded recombinant protein (Uxe1,