## 4 Metabolic Pathway and Host Engineering for Advanced Biofuels Production
### Advanced Biofuels Production from LCB-Derived Sugars or Hydrolysates  
Biofuel production from LCB has been primarily limited by pretreatment and saccharification of LCB, which determines sugar yields, and by inefficient co-utilization of hexose and pentose of host strains. While ethanol production has been more frequently chosen as a representative pathway to demonstrate biofuel production from LCB-derived sugars, a few studies on advanced biofuels production from LCB-derived sugars have been pursued recently [84]. One of the early studies demonstrated the engineering of _E. coli_ as a microbial factory for consolidated bioprocesses (CBP) by expressing enzymes required for both biomass degradation (cellulase, xylanase, \(\beta\)-glucosidase, and xylobiosidase) and biofuel synthesis (FAEE, butanol and pinene) [68]. Using this engineered _E. coli_ strain, 71 mg/L of FAEE, 28 mg/L butanol, and 1.7 mg/L pinene were produced from 5.5%, 3.3% and 3.9% _w/v_ IL-treated switchgrass, respectively. Although these titers need to be further improved for industrial application, it should be noted that 71 mg/L of FAEE was 80% of the estimated yield from 5.5% switchgrass that could release only 0.14% glucose and 0.14% xylose by the cellulose and the xylanase expressed from the engineered _E. coli_ strain [68]. A recent study demonstrated simultaneous isopentenol fermentation and saccharification of ionic liquid (IL)-pretreated pellets containing a mixture of four feedstocks [85]. The IL-pretreated pellet released 7 g/L glucose in 48 h, and almost 1 g/L of isopentenol was produced out of this hydrolysate. In another study, _E. coli_ was engineered to produce isobutanol from xylose, by integrating the isobutanol synthetic pathway and xylose utilization genes into the genome and using xylose as an inducer for the expression of these genes [86]. In this study, a titer of 3.6 g/L isobutanol was produced from cedar hydrolyses containing 86.4 g/L glucose and 15.5 g/L xylose although the productivity was 4.5 times lower than media containing pure glucose and xylose [86]. In addition, higher alcohols have been produced in _Corynebacterium crenatum_ via keto acid pathways using acid-pretreated hydrolyses of duckweed [87]. In this work, heterologous genes involved in isoleucine, leucine and valine biosynthesis pathways from _S. cerevisiae_ were expressed in _C. creanatum_, and 982 mg/L of 2-methyl-1-butanol, \(\sim\)1.1 g/L isobutanol and \(\sim\)685 mg/L of 3-methyl-1-butanol were produced from acid-pretreated hydrolyses of duckweed containing60 g/L glucose without compromising productivity [87]. Although raw hydrolysates were not used as a carbon source, Avicel hydrolysates containing cellobionic acid, one of the major components of lignocellulosic biomass, were also used to produce 1.4 g/L of isobutanol achieving 36% of the theoretical maximum with a productivity of 0.03 g/L/h by expressing a native gene, _ascB_ encoding 6-phospho-\(\mathfrak{p}\)-glucosidase [88]. Even though most studies showed that productivity was relatively reduced when biofuels were produced from LCB-derived hydrolysates or sugars, these results suggested that production of advanced biofuels from LCB-derived sugars is currently feasible, and it could be further improved by overcoming limitations that are not intrinsic to the engineered biofuel pathways and by further optimization of the responsible metabolic pathways.