An Ideal Biofuel Producing Microorganism (IBPM) should possess four important traits: it should be able to carry out (1) biomass degradation and (2) product formation; (3) it should show tolerance to solvents, and (4) it should serve as a chassis organism for rapid growth in the bioreactor (French 2009). Chassis organisms, such as yeast and E. coli, are well characterized. Commercial bioethanol has been produced from sugarcane by yeast. In addition, E. coli and Z. mobilis are progressing as efficient ethanol producers. A current challenge is to engineer biomass degradation (cellulolytic) ability. Further, investigators seek to enhance tolerance to harsh conditions that arise during cellulose fermentation, such as substrate and product toxicity. In particular, the chassis organism should have enhanced tolerance to toxic compounds of lignin. Classical strain improvement through long-term adaptation and mutagenesis may be an effective way to increase the tolerance to harsh environments, such as ethanol or lignin, because the mechanisms of toxicity and tolerance are largely unknown (Fischer et al. 2008).

Engineering cellulolytic ability into recombinant hosts has long been a challenge. The number of cellulase genes that should be cloned into the recombinant host remains unclear (Vinuselvi et al. 2011). The main obstacle to developing a recombinant cellulolytic host is the inability of hosts to support expression and secretion of a sufficient quantity of cellulases. Although cellulase expression is well established in yeast, there is no known study demonstrating direct conversion of plant biomass into ethanol. Despite the characterization of several cellulases in E. coli, a cellulolytic cassette containing all three cellulases has not been established for E. coli. Furthermore, efficient genetic tools are still lacking for Zymomonas, limiting its potential to be engineered with a heterologous gene.

One way to address the problems associated with heterologous cellulase expression and to reduce the metabolic burden imposed by the expression of cellulolytic enzymes in recombinant hosts would be the development of a well-defined synthetic consortium with two efficient players — native cellulolytic and solventogenic organisms —acting together. A high level of expression of multiple heterologous proteins would impose a heavy metabolic burden on the host. With a synthetic consortium, this burden could be shared by different species or by different strains of the same species. A co-culture of these strains to produce a cellulase cocktail would, therefore, reduces the overall metabolic burden and increase the ethanol yield (Brenner et al. 2008). Synthetic biology also offers superior inducible systems, such as light-inducible promoters and the fim inversion system, which are capable of providing spatiotemporal changes in gene expression (Levskaya et al. 2005; Ham et al. 2006). With such systems, it is possible to regulate the expression of genes with time and, potentially, to help reduce the metabolic burden imposed on the recombinant host (Drepper et al. 2011). Using metabolic engineering and synthetic biology, Steen et al. (2010) have developed a promising way of causing E. coli to produce more complex biofuels — fatty esters and fatty alcohols — directly from hemicellulose, a major component of plant-derived biomass (Fig 6). This study is representative of the recent progress in cellulosic fuel production. However, the possibility of increasing the productivity of such advanced biofuels remains a significant challenge.

5. Conclusions

Cellulosic bioethanol is gaining importance to circumvent the oil crisis and climate change. However, two major problems remain to be solved, in order to produce cellulosic ethanol economically. One problem is the high price of the cellulolytic enzymes used in the saccharification of lignocelluloses. The other problem is that the traditional saccharification and fermentation for bioethanol requires huge initial capital investment and operational cost. Consolidated bioprocessing presents a desirable way to produce bioethanol economically from lignocellulose. Microorganisms such as Trichoderma spp. and C. thermocellum effectively challenge the recalcitrance of lignocellulose, whereas microbes such as yeast and Z. mobilis can produce ethanol more efficiently. Several attempts have been made to combine these two abilities into a single organism, but with little success. Recent progress in synthetic biology, metabolic engineering, and protein engineering gives hope that the goal of generating cellulosic ethanol with a single organism may not be far from reality.

6. Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) through grants funded by the Ministry of Education, Science and Technology (NRF-2009- C1AAA001-2009-0093479, NRF-2009-0076912, NRF-2010-0006436) and UNIST (Ulsan National Institute of Science and Technology) research grant.