# 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
Using the XR-XDH pathway is likely to cause redox imbalance, and numerous studies have attempted to alleviate the cofactor imbalance by altering the cofactor specificity of the enzyme [78; 79; 80]. [PERSON]'s group [55] adjusted the copy numbers of three genes (_XYL1_, _XYL2_, and _XYL3_) in _S. cerevisiae_ to improve redox balance and reduce acetate and xylitol accumulation. These results of evolutionary engineering confirmed the importance of deleting _PHO13_ for xylose utilization. The final isobutanol titer was 2.6 g/L when coupled with the mitochondrial isobutanol pathway [56]. Metabolite analysis of the engineered strain revealed that the use of xylose increased valine levels in _S. cerevisiae_, which can be converted by branched-chain amino acid transaminases into KIV. This once again confirms that xylose promotes isobutanol synthesis. _C. glutamicum_ is also a promising industrial producer of isobutanol, and it is necessary to develop an engineered strain that can utilize cellulose. By heterologous expression the xylose XI and the arabinose metabolic pathways from _E. coli_, [PERSON]'s group [57] succeeded in constructing strains that can rapidly utilize mixed sugars, including glucose. When combined with previous strategies to optimize isobutanol synthesis [25; 58], 0.53 g/L of isobutanol could be produced from hemicellulose fractions. One way to address the fermentation of mixed sugars containing xylose is to bypass the use of glucose. Cellobiose is an intermediate product of cellulose hydrolysis to glucose and has no carbon catabolite repression effects on cells. Mixed sugar fermentation using cellobiose and xylose has been reported [81; 82], but no studies have addressed isobutanol synthesis. Isobutanol production using cellobiose was attempted based on a previously engineered strain from _E. coli_. Expression of \(\beta\)-glucosidase from _Thermobifida fusca_ resulted in an isobutanol production of 7.64 g/L by optimizing gene copy number [59]. It should be noted that direct transport of cellobiose to intracellular hydrolysis is required to avoid carbon catabolite repression, and no growth of cells expressing the cytoplasmic enzyme was observed in this study. Another study [60] tested cellobionic acid, the main product of cellulose hydrolysis assisted by the use of lytic polysaccharide monooxygenase (LPMO), as a carbon source for the production of isobutanol. _E. coli_ was found a naturally utilize the cellobionic acid pathway. A final titer of 1.4 g/L was produced from the cellulose hydrolysate of _Neurospora crassa_ conversion, thus expanding the available carbon source for cellulosic isobutanol. Most of the studies described above are based on either industrially produced cellulose or the monosaccharides obtained from its hydrolysis as substrates, but there is a lack of studies on the direct use of cellulose or its hydrolysates to produce isobutanol. Depending on the source of cellulosic biomass, the proportions of cellulose, hemicellulose, and lignin can vary considerably [83]. The hydrolysis products will also be significantly different, depending on how the pretreatment is metabolized [84]. These hydrolysis products (weak acids, furan derivatives, phenolic compounds, etc.) toxic to cells and reduce the isobutanol titer. Several experiments have tested the ability of engineered isobutanol-producing strains designed to ferment the hydrolysis products of cellulosic biomass. Duckweed [61; 85] is a fast-growing non-food crop with few growth requirements that is very easy to handle. Empty fruit bunches [62] are a large byproduct of palm oil production and sugarcane bagasse [63] is a common industrial byproduct. Their pretreated fermentation inhibitor content is low, as can be concluded from the comparison with glucose fermentation. This resulted in these biomasses being good carbon source providers. The best isobutanol titer was obtained from _Enterobacter aerogenes_[64] use of sugarcane bagasse hydrolysates to produce 23 g/L of isobutanol by pervaporation-coupled fermentation.