# Glucose Isomerase: Functions, Structures, and Applications
## 4 Application of GI
### Ethanol Production  
The bioconversion of a renewable biomass into ethanol is attractive in view of the rapid depletion of fossil fuels [62]. GI can catalyze the isomerization of xylose derived from hemicellulose biomass to xylulose, which can be fermented to ethanol by conventional yeast such as _Saccharomyces cerevisiae_, _Schizosaccharomyces pombe_, and _Candida tropicalis_[63; 64; 65]. This property of GI is attractive for the application in bioethanol production because no coenzyme is required and no intermediates are produced during the reaction [66]. However, the typical ethanol production process is plagued by low efficiency, owing to the low conversion yield of xylose to ethanol [67]. The GI enzyme engineering and strain improvement approaches have been explored to improve the conversion yield of xylose to ethanol [1; 65; 68]. The engineering of yeast strains that exhibit faster xylose metabolism is important in pursuing strain improvement for bioethanol production [69; 70].  
[PERSON] et al. evaluated the performance of SXA-R2P-E, an engineered isomerase-based xylose-utilizing _S. cerevisiae_ strain, for co-fermentation of xylose and glucose to ethanol [65]. SXA-R2P-E produced 50g/L ethanol (yield of 0.43 g ethanol/g sugar) in 72 h via a high-sugar fermentation process (70 g/L glucose and 40 g/L xylose). This strain also produced 18-21 g/L ethanol (yield of 0.43-0.46 g ethanol/g sugar) from acid-pretreated lignocellulosic hydrolysates such as rice straw and hardwood [65].  
[PERSON] et al. reported improved xylose fermentation through XI expression in the xylose-utilizing _S. cerevisiae_ strain IR-2, which has a deletion of the _GR3_ gene (encoding an endogenous xylose reductase) and overexpressed _XXE1_ (encoding an additional xylulose kinase). Evolutionary engineering was performed in the IR-2 strain to select high-efficiency XIs from eight previously reported XIs derived from various species [68]: _Burkholderia encuocepacia_ J2315 [71], _Lachoclostridium phytofermentans ISDg_ (LpXI) [67], _Orpinomyces_ sp. ukk1 [72], _Piromyces_ sp. E2 [73], _Prevotella ruminicola_ TC2-24 [74], _Ruminiclostridium cellulolyticum_ H10 [75], _Ruminococcus flaefaciens_ 17 [76], and _S. rubiginosus_[77]. Among them, the strain expressing LpXI exhibited the highest D-xylose consumption rate after 72 h of micro-aerobic fermentation on D-glucose and D-xylose mixed medium [68]. Furthermore, to enhance the LpXI catalytic activity, the authors performed random mutagenesis using an error-prone polymerase chain reaction and obtained two enzyme constructs with improved fermentation performances [68]. The SS120 strain, expressing double mutant LpXI (T63I/V162A), produced 53.3 g/L ethanol in 72 h (ethanol yield of approximately 0.44 (g/g-input sugars) from 85 g/L D-glucose and 35 g/L D-xylose [68].  
XI enzyme engineering is another approach that has been employed to increase the efficiency of bioethanol production by accelerating xylose metabolism [68; 78]. For example, the XI from the fungal strain _Piromyces_ sp. E2 (PirXI) is employed for xylose isomerization in engineered _S. cerevisiae_ strains but has low activity in vivo. To improve the performance of PirXI, [PERSON] et al. constructed a mutant library by substituting residues around the substrate and metal-binding sites of the enzyme [35]. PirXI variants were obtained by transferring the library to _S. cerevisiae_, followed by in vivo selection for enhanced xylose-supported growth under aerobic and anaerobic conditions. In particular, in the presence of Mg\({}^{2+}\) or Mn\({}^{2+}\), the K\({}_{\text{m}}\) of PirXI-V270A/A273G variant for xylose was higher, and the k\({}_{\text{cat}}\) was lower than the corresponding values for wild-type PirXI [35].  
Thus, further efforts to engineer GI/XI for a high xylose conversion rate through rational engineering or directed evolution are still needed, and applied research should be performed through in vivo studies on improved strains to enable efficient bioethanol production.