Contrary to rational engineering, partial and/or addi­tional metabolic pathways of microorganisms can be engineered to enhance bioproduct production. The term "metabolic engineering" was first coined by Bailey and was described as a vast variety of manipulations and experimental procedures to improve the productiv­ity of a desired metabolite by an organism (Bailey, 1991). More specifically, examples of metabolic engineering can include increased productivity and/or yield, improvement of substrate uptake, widening the scope of substrate range for an organism, modification of metabolic flux, and elimination of unnecessary or competing metabolic pathways (Stephanopoulos, 1999).

Metabolic engineering, similar to rational engineer­ing, requires the selection of a good host/microor — ganism as a candidate for the production of biofuels and/or bioproducts from biomass. This could include engineering desired pathways into well-studied host microorganisms such as Escherichia coli and Saccharo — myces cerevisiae; these microorganisms have been used for industrial-scale production for several years. How­ever, some experts suggest that engineering desired pathways into microorganisms that already possess industrial properties may be more successful. This is due to the potential for metabolic burden to the cell; new metabolic pathways require amino acids, redox cofactors, and energy for synthesis and function of its enzymes (Lee et al., 2008a).

Furthermore, metabolic engineering poses several general challenges for researchers including the devel­opment of recombinant DNA technologies for selected host microorganisms, development of quantitative tools, methods to understand flux modification in complex biological systems, and the development of quantitative techniques to determine changes in fluxes or metabolite concentrations (Cameron and Tong, 1993). A few suc­cessful examples of metabolic engineering to improve general host and select host microorganisms metabolism for the digestion and conversion of biomass are outlined below.

Recently, the development of genome-scale modeling permits the prediction of how new metabolic pathways may impact growth and product production using meta­bolic models. These models result in a more rational approach to metabolic engineering (Patil et al., 2004). Moreover, stoichiometric models can be defined by established equations through the use of metabolic flux analysis (MFA); this is established by measuring ex­change fluxes experimentally (Lee et al., 2008b). For example, the native metabolism of E. coli under different growth conditions (Kayser et al., 2005) and during re­combinant protein production (Ozkan et al., 2005) has been determined using MFA. For efficient application in biofuel and bioproduct production, genome-scale models should be developed with constraints to opti­mize flux in desired pathways, while balancing impor­tant cofactors and energy metabolites (Lee et al., 2008b).

Host microorganisms such as E. coli and S. cerevisae have been improved time and again for the fermentation of sugars to ethanol. In particular, due to the broad range of carbohydrates metabolized by E. coli, it has been a po­tential candidate for the expression of ethanologenic pathways in some studies. For example, a portable cassette called the production of ethanol operon (PET operon) was used to genetically engineer the homoetha — nologenic pathway from Zymomonas mobilis into E. coli, which included the pyruvate decarboxylase and alcohol dehydrogenase B genes. Using the PET system, these genes were integrated into the chromosome of E. coli at the pfl locus. Meanwhile the fumarate reductase (frd) gene was deleted to eliminate succinate production, therefore preventing carbon loss. These metabolic changes resulted in the recombinant strain KO11, which produced ethanol yields as high as 95% in complex me­dium (Jarboe et al., 2007; Ohta et al., 1991). However, host strains such as E. coli may encounter metabolic bur­dens and are often not naturally adapted to the toxicity of end products like ethanol. Thus, there have also been some attempts to metabolically engineer known biomass-converting bacteria or fungal strains.

Typically, bacteria produce more desirable end prod­ucts through facultative and anaerobic digestion, as is the case for bacteria belonging to the class Clostridia. Much of the metabolic engineering in these species fo­cuses on product formation, which may include the elimination of undesirable products such as in the case of an engineering project conducted on Clostridium acetobutylicum—a well-known ethanogenic strain stud­ied often for the production of butanol. In brief, the ace- toacetate decarboxylase gene (adc) was disrupted in the hyperbutanol-producing strain C. acetobutylicum EA 2018 using TargeTron technology (Sigma Aldrich) (Jiang et al., 2009). TargeTron is a group II intron developed for rapid and site-specific gene disruption in prokaryotes. The disruption of adc led to an increase in butanol ratio from 70% to 80.05%, with a simultaneous reduction in acetone of 0.21 g/l (Jiang et al., 2009).

In contrast, one can implement metabolic engineering to improve native metabolism in microorganisms by engineering entirely novel pathways for desired product formation, which is more practically done in hosts able to hydrolyze biomass, such as the example with Clos­tridium cellulolyticum. Recently, Higashide et al. demon­strated the production of isobutanol from crystalline cellulose in C. cellulolyticum (Higashide et al., 2011). In this study, the development of valine biosynthesis pathway required the expression of five genes, alsS, ilvC, ilvD, kivD, and ahdA, to convert pyruvate into iso­butanol. Consequently, only the expression and function of kivD (2-keto-acid decarboxylase) and alsS (alpha — acetolactate synthase) were confirmed; nonetheless modified C. cellulolyticum produced up to 660 mg/l of isobutanol over a 7- to 9-day growth period (Higashide et al., 2011).

These examples of engineering and modeling to improve the metabolic capabilities of strains helped lay the foundation for future development of biomass­converting microorganisms. Combined with the ability to rationally design enzymes with greater stability and/or increased specific activity the modification of microorganisms in industrial production of biofuels and bioproducts looks promising.