## 3 Metabolic Pathway and Host Engineering for Ethanol Production  
Since sugars are both carbon and energy sources for biomass and ethanol production, more efficient uptake and utilization of various sugars are important factors that can improve ethanol productivity. For example, the uptake rate of sucrose was improved in yeast for more efficient utilization of sucrose from sugarcane [12]. In this study, a _S. cerevisiae_ strain with intracellularly localized sucrose invertase (iSUC1) was evolved in sucrose-limited chemostat, and the evolved strain showed higher sucrose-proton symporter activity and increased ethanol yield by 11% [12]. In general, most industrial microbial hosts have a good capability of utilizing hexose, but restricted capability in utilizing pentose such as xylose, the second most abundant sugar in biomass, and arabinose due to lack of the pentose utilization pathway and a catabolite repression in the presence of glucose. Although there have been efforts to expand the substrate utilization capability of yeast by engineering the substrate affinity of sugar transporters towards pentose rather than hexose, no significant progress has been made yet [13]. In one study, transporter mutants of GXS1 from _Candida intermedia_ and XUT3 from _Scheffersomyces stipitis_ were expressed, and it showed an increased growth rate of yeast on xylose by 70% and a changed pattern of diauxic shifts [14]. In a following study, [PERSON] and colleagues identified a sequence motif  
Figure 2: Ethanol fermentation pathways in _S. cerevisiae_ (solid line, EMP glycolysis pathway) and _Z. mobilis_ (dashed line, ED glycolysis pathway). While the EMP pathway produces two ATPs per glucose molecule, the ED pathway produces only one ATP molecule per glucose molecule. KDPG, 2-keto-3-deoxy-6-phosphogluconate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-diphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate. of G-G/F-XXX-G, of which saturation mutagenesis generated transporter mutants that have an exclusive specificity for xylose but not for glucose. All these mutants, however, were still found to be repressed by glucose [15]. In another study, [PERSON] and colleagues [16] screened glucose-insensitive xylose transporter mutants. In this study, they found a mutation at either of two conserved residues located near the entrance of sugar-binding pocket, and a mutant of a yeast hexose transporter (Gal2-N376F) was identified to have the highest affinity for xylose among mutants without glucose transport activity [16]. Another metabolic engineering strategy is to maximize the flux of sugars to ethanol while minimizing the flux to biomass or to other fermentation byproducts such as glycerol. Since formation of highly reduced fermentation products such as glycerol is driven by accumulation of cytosolic NADH, _S. cerevisiae_ have been engineered to maintain lower level of cytosolic NADH by various genetic modifications such as the deletion of NADPH-dependent glutamate dehydrogenase (GDH1) along with the overexpression of glutamate-ammonia ligase (GLN1) and glutamate transporter (GLT1); and substitution of innate glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with heterologous GAPDH from either _Bacillus cereus_ or _Streptococcus mutans_. The latter approach aimed to decrease cytosolic NADH formation and ATP production by using an alternative non-phosphorylating, NADP\({}^{+}\)-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN) of GAPDH [13]. In addition to the resorts to decrease cytosolic NADH, metabolic pathways of ethanologenic _E. coli_ were redesigned at the systems level by elementary mode analysis for metabolic coupling of biomass and ethanol production. Deletion of 9 genes in central metabolism was suggested by elementary mode analysis, and the engineered _E. coli_ strain produced 90% of theoretical yield after 48 h fermentation [17].