# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 3 Biomass Isobutanol Production
### Isobutanol Production from Lignocellulose
#### 3.1.2 Cellulosic Isobutanol Produced by Non-Native Cellulose-Degrading Microorganisms  
Another strategy for obtaining cellulosic isobutanol is to further reform the pre-existing engineered strains for isobutanol production to utilize lignocellulosic or its pretreatment products. _E. coli_ and _S. cerevisiae_ are the most promising cellulosic isobutanol-producing strains. Two model organisms have invested considerable research in cellulose utilization, which have been summarized in a considerable number of reviews [74; 75]. They both have significant carbon catabolite repression and cannot utilize glucose simultaneously with other sugars. Xylose is the most abundant sugar in lignocellulosic aside from glucose, so many efforts have been to construct industrially viable xylose-utilizing strains. To eliminate carbon catabolite repression, _E. coli_ used UV mutagenized screened a new target gene [49], _MLc_, encoding a DNA-binding transcriptional repressor. A strain with this mutant _MLc_ gene outperformed previous research in fermenting isobutanol from mixed glucose and xylose. Further optimization of the isobutanol synthesis pathway genes expression (_ilv_C, _ilvD_, _alsS_, _kivd_ and _adhA_) with the biomass-inducible chromosome-based expression system (BICES) [50], achieved an isobutanol titer of 3.7 g/L from cedar hydrolysate. _S. cerevisiae_ cannot convert xylose naturally, but considerable efforts have been made to engineer a strain of _S. cerevisiae_ that can [76]. There are two types of xylose metabolism (Figure 1D), one catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH), the other by xylose isomerase (XI). However, the XR-XDH pathway requires the cofactor NADPH that is also in high demand for isobutanol synthesis, prone to cofactor imbalance. For this reason, the XI pathway is favored for isobutanol synthesis, but most attempts to express heterologous xylose isomerases in _S. cerevisiae_ have failed. XylA, a xylose isomerase from _Clostridium phytofermetants_, was successfully expressed in _S. cerevisiae_ by [PERSON] group [51]. Further, they increased xylose metabolism by overexpression _Xks1_ and _Tal1_, with the redesigned cytoplasmic isobutanol pathway, it achieved first isobutanol production by using xylose as the sole carbon source. [PERSON] group engineered a strain to produce ethanol from xylose by the introducing xylose isomerase from _Pioromyces sp._ E2 in _S. cerevisiae_. Overexpression _RK11_, _RPE1_, _TKL1_, _TAL1_ and _XYL3_ in this background increased xylose assimilation and facilitated adaptive laboratory evolution [52]. [PERSON] group [53] further optimized the use of xylose in this strain by knocking out gene _PHO13_. With expression of the mitochondrial isobutanol pathway and inhibition of valine and acetic acid synthesis, the production of isobutanol was 3.1 g/L in 192 h of fed-batch fermentation. These experiments show that xylose promotes mitochondrial activity significantly more than glucose does and increases with increasing concentrations of xylose. Thus, the advantages of isobutanol production through the mitochondrial isobutanol pathway from xylose were demonstrated. Another xylose utilization pathway, the XR-XDH pathway, has also been used to produce isobutanol. [PERSON]'s group [77] determined through combinatorial screening that the xylose metabolism pathway from _Scheffersomyces stipitis_ is most effective when applied to isobutanol synthesis. Incorporating the strategies identified in previous studies to enhance xylose conversion and assimilation (knockout _PHO13_, _GRE3_ and overexpression of _XYL3_) and optimizing the copy number of isobutanol pathway genes produced 48.4 mg/L of isobutanol within 144 h. Adaptive laboratory evolution of the resulting engineered strain, and identification of two newly discovered mutation targets (the _CCR46385_ and _TIFAF79_) to improve xylose utilization [54] increased the xylose ratio growth rate by 40.6%, but had little impact on isobutanol production.