The release of C5 and C6 sugars from lignocellulosic biomass deconstruction supports the growth of heterotrophic microorganisms and the metabolic conversion of sugars into biofuel. Representative hydrocarbon-based fuel titers produced by engineered, heterotrophic hosts are listed in Table 1. The most common heterotrophic hosts for biofuel production are the model organisms Escherichia coli and Saccharomyces cerevisiae. These hosts are attractive candidates for fuel production due to their fast growth rates, well-known genetics and regulation, advanced molecular tools for genetic engineering, and established use in the industrial setting. Neither E. coli nor S. cerevisiae naturally produce significant amounts of hydrocarbon-based fuels, necessitating the application of metabolic engineering techniques. Heterotrophic organisms that naturally produce hydrocarbon-based fuels are also potential hosts for large-scale biofuel production. For example, Bacillus subtilis naturally produces higher concentrations of isoprene than other commonly known bacteria like E. coli [55]. B. subtilis is also a model organism for Gram-positive bacteria with established tools for genetic modification, advancing its appeal as a host for isoprene production. Similarly, heterotrophic algae can produce significant quantities of TAG. This has motivated some preliminary investigation into engineering the model green alga, Chlamydomonas reinhardtii, for TAG production [56—58]. While most meta­bolic engineering efforts have focused on these model heterotrophic hosts, genetic tools can be developed for other organisms with desirable fuel production traits.

Most heterotrophic hosts for biofuel production utilize the Embden-Meyerhof-Parnas (EMP) pathway for sugar catabolism (Figure 4). The EMP pathway has evolved for efficient carbon utilization and is typically not rate-limiting for fuel production. As such, EMP pathway enzymes are not often targeted for genetic manipulation. However, the organic feedstock from lignocellulose deconstruction is comprised of a range of sugars, including hexoses: glucose, mannose, and galactose, and pentoses: xylose and arabinose [79]. A major concern in convert­ing these sugars into fuel is the efficient utilization of all available hexoses and pentoses. While some organisms like E. coli can naturally metabolize these different forms of sugar, others, like S. cerevisiae, can only utilize specific forms [80]. S. cerevisiae does not naturally express path­ways for catabolizing pentoses. There are two known pathways for xylose catabolism, both of which have been expressed in S. cerevisiae [81—83]. Xylose can be converted into xylulose-5- phosphate (Xu5P), an intermediate in the pentose phosphate pathway (PPP), through expres­sion of a xylose isomerase (XI) and xylulose kinase (XK) [82]. Alternatively, the XI can be replaced by a xylose reductase (XR) and xylitol dehydrogenase (XDH) [81, 82]. Complications

in these two xylose utilization pathways include the inhibition of XI by xylitol (Xol) and the reducing equivalents required by XR and XDH [80]. Successful strategies for engineering xylose utilization in S. cerevisiae include expression of a fungal XI from Piromyces sp. E2 along with overexpression of the non-oxidative PPP pathway [84] and expression of XR and XDH from the xylose-fermenting yeast Pichia stipitis [85]. Two pathways have also been expressed in S. cerevisiae for arabinose utilization [86, 87]. The bacterial pathway for arabinose catabolism consists of 3 enzymatic steps, while the fungal pathway involves 5 enzymatic steps, 4 of which require cofactors of NADPH or NAD+ (Figure 4). Efficient arabinose utilization in S. cerevi — siae has been achieved through heterologous expression of a bacterial arabinose catabolism pathway along with overexpression of the non-oxidative PPP and evolutionary engineering [88]. While most of these metabolic engineering examples focus on utilizing sugars for fermentation to ethanol, the strategies for engineering carbon utilization can also be applied for hydrocarbon-based fuel production.

Unlike S. cerevisiae, E. coli can utilize the hexoses and pentoses derived from lignocellulose; however, the carbon catabolite repression (CCR) system in E. coli leads to inefficient, diauxic growth [89]. Through CCR, E. coli sequentially consumes different sources of organic carbon based on substrate preference, leading to delayed and often incomplete utilization of unpre­ferred sugars like xylose and arabinose. This translates into lower productivities and yields along with downstream complications due to the presence of unmetabolized sugars [80]. As a result, CCR is often targeted by metabolic engineering to alleviate these undesired effects. A common engineering strategy is to use mutants of the transcriptional activator CRP (cyclic AMP receptor protein) which have been modified to eliminate the allosteric requirement for cAMP, thereby leading to expression of the pentose catabolizing pathways in the presence of the preferred substrate, glucose [90]. The phosphotransferase system (PTS), responsible for the preferential uptake of glucose, has also been deleted to encourage simultaneous utilization of mixed sugars [91]. Lastly, deletion of methylglyoxyal synthase was shown to improve the co­metabolism of sugars, ostensibly due to elimination of methylglyoxyal, an inhibitor of sugar metabolism [92]. Through modifying the components of CCR, E. coli can be engineered to efficiently utilize the organic carbon mixture resulting from lignocellulose degradation.

In addition to the hexoses and pentoses derived from lignocellulosic biomass, glycerol may soon become an inexpensive organic carbon source for fuel production. Glycerol is a byproduct of the conversion of TAG into biodiesel during algal biofuel processing, and thus, large quantities of glycerol may be available for use as an organic carbon source. The main pathway for aerobic glycerol utilization involves a two-step conversion to produce the glycolytic metabolite DHAP [93]. The glycerol utilization pathway is not a common target for metabolic engineering, yet glycerol has been reported as a supplementary carbon source for the produc­tion of isoprenoid-based fuels, farnesol and a-farnesene [47, 48]. Future metabolic engineering efforts may focus more on glycerol utilization as the availability of glycerol increases.

Second generation biofuel production still remains to be demonstrated at large scales, yet the overall process is easily integrated with current technologies. Equipment and practices used for agricultural harvesting can be directly applied to harvesting lignocellulosic biomass. In fact, some agricultural processes already produce biomass waste streams that can be utilized for feedstock, such as corn stover. Moreover, commercial fermenters can be employed as bioreactors for the microbial fuel conversion. The main technical difficulties in large-scale lignocellulosic fuel production center on provision of the carbon source. The quantities of biomass needed to support industrial-scale fuel production will require a significant invest­ment of land and nutrient resources, and the supply will be subject to varying climate conditions. A supply chain infrastructure must also be constructed to harvest the biomass and transport it to the production facilities. A primary technical focus of current research on lignocellulosic-derived fuels is the deconstruction of biomass into useable sugars. The thermal, chemical, and enzymatic processes for biomass deconstruction have been a limiting factor for economical second generation biofuel production [94, 95]. As the cost of biomass deconstruc­tion is reduced with new technology, the large-scale production of second generation biofuels will begin to contribute to the world’s supply of renewable energy.